Use of armadillo repeat (arm1) polynucleotides for obtaining pathogen resistance in plants

ABSTRACT

The invention relates to a method of generating or increasing a pathogen resistance in plants by reducing the expression of at least one Armadillo repeat polypeptide or a functional equivalent thereof. The invention relates to novel nucleic acid sequences coding for a  Hordeum vulgare  Armadillo repeat (HvARM) polynucleotide and describes homologous sequences (ARM1) thereof, and to their use in methods for obtaining a pathogen resistance in plants e, and to nucleic acid constructs, expression cassettes and vectors which comprise these sequences and which are suitable for mediating a fungal resistance in plants. The invention furthermore relates to transgenic organisms, in particular plants, which are transformed with these expression cassettes or vectors, and to cultures, parts or transgenic propagation material derived therefrom.

The invention relates to a method of generating or increasing a pathogenresistance in plants by reducing the expression of at least oneArmadillo repeat polypeptide or a functional equivalent thereof. Theinvention relates to novel nucleic acid sequences coding for a Hordeumvulgare Armadillo repeat (HvARM) polynucleotide and describes homologoussequences (ARM1) thereof, and to their use in methods for obtaining apathogen resistance in plants, and to nucleic acid constructs,expression cassettes and vectors which comprise these sequences andwhich are suitable for mediating a fungal resistance in plants. Theinvention furthermore relates to transgenic organisms, in particularplants, which are transformed with these expression cassettes orvectors, and to cultures, parts or transgenic propagation materialderived therefrom.

There are only few approaches which confer a resistance to pathogens,mainly fungal pathogens, to plants. This shortcoming can partly beattributed to the complexity of the biological systems in question.Another fact which stands in the way of obtaining resistances topathogens is that little is known about the interactions betweenpathogen and plant. The large number of different pathogens, theinfection mechanisms developed by these organisms and the defensemechanisms developed by the plant phyla, families and species interactwith one another in many different ways.

Fungal pathogens have developed essentially two infection strategies.Some fungi enter into the host tissue via the stomata (for examplerusts, Septoria species, Fusarium species) and penetrate the mesophylltissue, while others penetrate via the cuticles into the epidermal cellsunderneath (for example Blumeria species).

The infections caused by the fungal pathogens lead to the activation ofthe plants defense mechanisms in the infected plants. Thus, it has beenpossible to demonstrate that defense reactions againstepidermis-penetrating fungi frequently start with the formation of apenetration resistance (formation of papillae, strengthening of the cellwall with callose as the main constituent) underneath the fungalpenetration hypha (Elliott et al. Mol Plant Microbe Interact. 15:1069-77; 2002).

In some cases, however, the plant's defense mechanisms only confer aninsufficient protection mechanism against the attack by pathogens.

The formation of a penetration resistance to pathogens whose infectionmechanism comprises a penetration of the epidermal cells or of themesophyll cells is of great importance both for monocotyledonous and fordicotyledonous plants. In contrast to described mlo-mediated resistance,it can probably make possible the development of a broad-spectrumresistance against obligatory biotrophic, hemibiotrophic andnecrotrophic fungi.

The present invention was therefore based on the object of providing amethod for generating a resistance of plants to penetrating pathogens.

The object is achieved by the embodiments characterized in the claims.

The invention therefore relates to a method of increasing the resistanceto one or more penetrating pathogen(s) in a monocotyledonous ordicotyledonous plant, or a part of a plant, for example in an organ,tissue, a cell or a part of a plant cell, for example in an organelle,which comprises lessening or reducing the activity or amount of anArmadillo repeat ARM1 protein in the plant, or a part of the plant, forexample in an organ, tissue, a cell or a part of a cell, for example ina cell compartment, for example in an organelle, in comparison with acontrol plant or a part of a control plant, for example its organ,tissue, cell or part of a cell, for example in a cell compartment, forexample in an organelle.

Preferably, a race-unspecific resistance is obtained in the methodaccording to the invention. Thus, for example, a broad-spectrumresistance against obligatorily biotrophic and/or hemibiotrophic and/ornecrotrophic fungi of plants, in particular against mesophyll, epidermisor mesophyll-penetrating pathogens, can be obtained by the methodaccording to the invention.

Surprisingly, it has been observed that the gene silencing by means ofdsRNAi of a gene which codes for an Armadillo repeat protein HvARM ofbarley results in an increase in the resistance of monocotyledonous anddicotyledonous plants to fungal pathogens. Thus, this negative controlfunction in the event of attack by fungal pathogens has beendemonstrated for the Armadillo repeat ARM1 protein from barley (Hordeumvulgare) (HvARM1), wheat (Triticum aestivum) and thale cress(Arabidopsis thaliana).

It has been determined within the scope of a TIGS (=Transient InducedGene Silencing) analysis in barley by the method of Schweizer et al.(Plant J. 2000 December; 24(6): 895-903) that a dsRNAi-mediatedsilencing of the gene HvARM greatly increases the resistance to Blumeriagraminis f. sp. hordei (synonym: Erysiphe graminis DC. f. sp. hordei).This effect has also been obtained in dicotyledonous species such as,for example, Arabidopsis thaliana by inducing the post-transcriptionalgene silencing (PTGS). This emphasizes the universal importance of theloss-of-function of HvARM1-homologous genes for the development of abroad-spectrum pathogen resistance of the plant.

The Armadillo repeat motif was originally found in the Drosophilamelanogaster segment polarity gene armadillo. It codes for abeta-catenin which plays an important part in cell-cell adhesion and incell differentiation. Armadillo (Arm) repeat proteins comprise copiesarranged in tandem of a degenerated sequence of approx. 42 amino acids,which encodes a three dimensional structure for mediatingprotein-protein interactions (Azevedo et al. (2001) Trends Plant Sci. 6,354-358). Most of these proteins are involved in intracellular signaltransduction or in regulating gene expression within the framework ofcellular developmental processes. Contrary to the situation in animals,only two plant Armadillo repeat proteins have been functionallycharacterized: the first gene is potato PHOR1 (photoperiod-responsive 1)which was shown to play a part in gibberellic acid signal transduction(Amador V, Cell 10; 106(3):343-54.). The second Armadillo repeat proteinis oilseed rape ARC1 (Armadillo repeat-containing protein 1) whichinteracts with the SRK1 receptor kinase (Gu et al. (1998) Proc. Natl.Acad. Sci. USA 95, 382-387). It therefore plays an important part in theregulation of oilseed rape self-incompatibility. Transgenic plants whoseARC1 expression has been reduced by silencing exhibit reducedself-incompatibility. Interestingly, ARC1 belongs to the U-Boxcomprising subclass of Armadillo repeat proteins, which class includes18 genes in Arabidopsis (Azevedo et al. (2001) Trends Plant Sci. 6,354-358). The U-box is a motif comprising approx. 70 amino acidresidues. Besides the HECT and the RING Finger proteins they presumablyform a third class of ubiquitin E3 ligases whose primary function isthat of establishing substrate specificity of the ubiquitinationapparatus (Hatakeyama et al. (2001) J. Biol. Chem. 76, 33111-33120).

The genes or the nucleic acids used or the expressed proteins whoseexpression is reduced preferably have an identity of 40% or more,preferably 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or more,compared to the particular sequence of HvARM (SEQ ID NO: 1 and SEQ IDNO: 2). The genes with the highest homologies to HvArm, from rice (Acc.No.: XM_(—)479734.1, XM_(—)463544, AP003561, or XM_(—)506432), tobacco(AY219234) and Arabidopsis (Acc. No. NM_(—)127878, AC004401, BT020206,AB007645, NM_(—)115336, AK118613, AL138650, AL133314, AC010870,AY125543, AY087360, AB016888, AK175585, AL049655, AY096530 andAK118730), thus presumably carry out similar functions as HvARM in theplant. These are therefore included in the generic term “Armadillorepeat ARM1” or “ARM1” protein hereinbelow. In contrast, HvARM andHvARM1 refer to such a protein from barley.

Recently, another plant Armadillo repeat protein, SpI11 in corn, hasbeen described, for which a regulation of the plant cell death responsewithin the framework of abiotic stress response has been detected. Theloss of function of the corresponding gene results in a “lesion mimic”phenotype which impairs the agronomic performance of the plant (Zeng LR, (2004) Plant Cell. 16(10):2795-808). Interestingly, the sequencehomology of SpI11 to HvARM is only 23.4% at the amino acid level.Without being bound or limited by theory, the low sequence homology, inaddition to the different functions, also indicates that HvARM and SpI11belong to different subclasses of Armadillo repeat proteins.

Consequently, it came as a surprise that reducing HvARM1 gene expressionby RNAi-mediated silencing results in an increase in the resistance ofbarley to barley mildew and that this negative control function with anattack by fungal pathogens was likewise shown in wheat (Triticumaestivum) and thale cress (Arabidopsis thaliana).

In a further embodiment, the invention therefore relates to a method ofgenerating a plant with an increased resistance to one or more plantpathogen(s), preferably with a broad-spectrum resistance, in particularto fungal pathogens, for example from the classes Ascomycetes,Basidiomycetes, Chytridiomycetes or Oomycetes, preferably of mildews ofthe family Erysiphaceae, and particularly preferably of the genusBlumeria, that is by reducing expression of a protein which ischaracterized in that it comprises at least one Armadillo repeat. Theprotein preferably comprises two, particularly preferably more than two,Armadillo repeats.

In a further embodiment of the method of the invention, the activity ofan Armadillo repeat polypeptide is reduced, for example blocked oreliminated, which polypeptide essentially does not comprise a U-box,i.e. which does not comprise any U-box or any functional U-box.

In a further embodiment, in the method according to the invention theactivity of a polypeptide is reduced or eliminated, which is encoded bya polynucleotide comprising at least one nucleic acid molecule selectedfrom the group consisting of:

-   (a) nucleic acid molecule which codes for at least one polypeptide    comprising the sequence as shown in SEQ ID No: 2, 4, 6, 8, 10, 12,    14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 60,    61 or 62;-   (b) nucleic acid molecule which comprises at least one    polynucleotide of the sequence as shown in SEQ ID No: 1, 3, 5, 7, 9,    11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, or    43;-   (c) nucleic acid molecule which codes for a polypeptide whose    sequence has at least 50% identity to the sequences SEQ ID No: 2;-   (d) nucleic acid molecule according to (a) to (c) which codes for a    fragment or an epitope of the sequences as shown in SEQ. ID No.: 2,    4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,    40, 42, 44, 60, 61 or 62;-   (e) nucleic acid molecule which codes for a polypeptide which is    recognized by a monoclonal antibody directed against a polypeptide    which is encoded by the nucleic acid molecules as shown in (a) to    (c);-   (f) nucleic acid molecule which hybridizes under stringent    conditions with a nucleic acid molecule as shown in (a) to (c); and-   (g) nucleic acid molecule which can be isolated from a DNA library    using a nucleic acid molecule as shown in (a) to (c) or their    part-fragments of at least 15 nt, preferably 20 nt, 30 nt, 50 nt,    100 nt, 200 nt or 500 nt, as probe under stringent hybridization    conditions;    or comprises a complementary sequence thereof is reduced, for    example eliminated.

In the method according to the invention, it is in particular theresistance to mesophyll- and/or epidermis-cell-penetrating pathogenswhich is preferably increased.

In one embodiment, the resistance is obtained by lessening, reducing orblocking the expression of a polypeptide, preferably of a polypeptidewhich is encoded by the above-described nucleic acid molecule, forexample that of an ARM1 from rice (Acc. No.: XM_(—)479734.1,XM_(—)463544, AP003561, or XM_(—)506432), tobacco (AY219234) andArabidopsis (Acc. No. NM_(—)127878, AC004401, BT020206, AB007645,NM_(—)115336, AK118613, AL138650, AL133314, AC010870, AY125543,AY087360, AB016888, AK175585, AL049655, AY096530 and AK118730).

On the other hand, it is also possible to reduce, lessen or block theendogenous activity of one of these polypeptides by methods known to theskilled worker, for example by mutating a genomic coding region for theactive center, for binding sites, for localization signals, for domains,clusters and the like, such as, for example, of coding regions forcoiled coil, HEAT, FBOX, LRR, IBIB, C2, WD40, beach, U-box or UNDdomains. The activity can be reduced in accordance with the invention bymutations which affect the secondary, tertiary or quaternary structureof the protein.

Mutations can be inserted for example by an EMS mutagenesis. Domains canbe identified by suitable computer programs such as, for example, SMARTor InterPRO, for example as described in Andersen P., The Journal ofBiol. Chemistry, 279, 38, pp. 40053-40061, 2004 or Y. Mudgil, PlantPhysiology, 134, 59-66, 2004, and literature cited therein. The suitablemutants can then be identified for example by tilling (Henikoff et al.Plant Physiol. 2004 June; 135(2):630-6).

In another embodiment, the lessening of the polypeptide quantity,activity or function of an Armadillo repeat ARM1 protein in a plant iscombined with increasing the polypeptide quantity, activity or functionof other resistance factors, preferably of a Bax inhibitor 1 protein(BI-1), preferably of the Bax inhibitor 1 protein from Hordeum vulgare(GenBank Acc. No.: AJ290421), from Nicotiana tabacum (GenBank Acc. No.:AF390556), rice (GenBank Ace. No.: AB025926), Arabidopsis (GenBank Acc.No.: AB025927) or tobacco and oilseed rape (GenBank Acc. No.: AF390555,Bolduc N et al. (2003) Planta 216:377-386) or of ROR2 (for example frombarley (GenBank Acc. No.: AY246906), SnAP34 (for example from barley(GenBank Acc. No.: AY247208) and/or of the lumenal binding protein BiPfor example from rice (GenBank Acc. No. AF006825). An increase can beachieved for example by mutagenesis or overexpression of a transgene,inter alia.

In one embodiment, a lowering of the protein quantity or activity orfunction of the proteins RacB (for example from barley (GenBank Ace.No.: AJ344223)), CSL1 (for example from Arabidopsis (GenBank Acc. No.:NM116593), HvNaOX (for example from barley (GenBank Acc. No.: AJ251717),MLO (for example from barley (GenBank Acc. No. Z83834) is achieved.

The activity or function of MLO, BI-1 and/or NaOX can be reduced orinhibited analogously to what has been described for MLO in WO 98/04586;WO 00/01722; WO 99/47552 and the further publications mentionedhereinbelow, whose content is herewith expressly and expressis verbisincorporated by reference, in particular in order to describe theactivity and inhibition of MLO. The description of the abovementionedpublications describes processes, methods and especially preferredembodiments for lessening or inhibiting the activity or function of MLO;the examples indicate specifically how this can be realized.

The reduction of the activity or function and, if appropriate of theexpression of BI-1 is described in detail in WO 2003020939, which isherewith expressly and expressis verbis incorporated into the presentdescription. The description of the abovementioned publication describesprocesses and methods for lessening or inhibiting the activity orfunction of BI-1; the examples indicate specifically how this can berealized. The reduction or inhibition of the activity or function ofBI-1 is especially preferably carried out in accordance with theembodiments especially preferred in WO 2003020939 and the examples andin the organisms shown therein as being especially preferred, inparticular in a plant, for example constitutively, or in a part thereof,for example in a tissue, but especially at least in the epidermis or ina considerable part of the epidermal cells. The reduction of theactivity or function and, if appropriate of the expression, of BI-1 isdescribed extensively in WO 2003020939. The skilled worker finds in WO2003020939 the sequences which code for BI-1 proteins and can alsoidentify 31-1 with the method provided in WO 2003020939.

The reduction of the activity or function and, if appropriate of theexpression, of NaOX is described extensively in PCT/EP/03107589, whichis herewith expressly and expresses verbis incorporated into the presentdescription. The description of the abovementioned publication describesprocesses and methods for lessening or inhibiting the activity orfunction of NaOX, and the examples indicate specifically how this can berealized. The reduction or inhibition of the activity or function ofNaOX is especially preferably carried out in accordance with theembodiments especially preferred in PCT/EP/03/07589 and the examples andin the organisms shown therein as being especially preferred, inparticular in a plant, for example constitutively, or a part thereof forexample in a tissue, but especially advantageously at least in theepidermis or in a considerable part of the epidermal cells. The skilledworker finds in PCT/EP/03/07589 the sequences which code for NaOXproteins and can also identify NaOX with the method provided inPCT/EP/03/07589.

The terms “to lessen”, “to reduce” or “to repress” or their substantivesare used synonymously in the present text.

“Lessening”, “reduction” or “repression” or their verbs are understoodas meaning, in accordance with the invention, that the activity in theplant is lower than in a control plant or is lower in a part of a plantthan in a corresponding part of a control plant, for example in anorgan, an organelle, a tissue or a cell. In preferred embodiments, theactivity of the abovementioned polypeptide is 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, 97%, 99% or even lower than in the control. Inone embodiment, essentially no expression or particularly preferably noexpression at all of the abovementioned polypeptide takes place. As aconsequence, these terms also comprise the complete inhibition orblocking of an activity, for example by the knock-out of a gene.

“Reduction”, “to reduce”, “lessening” or “to lessen”, “repression” or“to repress” comprise the partial or essentially complete inhibition orblocking of the functionality of a protein, based on a variety ofcell-biological mechanisms.

Lessening within the purpose of the invention also comprises aquantitative reducing of a protein down to an essentially complete (i.e.lack of detectability of activity or function or lack of immunologicaldetectability of the protein) or complete absence of the protein. Here,the expression of a certain protein or the activity or function in acell or an organism is reduced by preferably more than 50%, especiallypreferably by more than 80%, and in particular by more than 90%.

In a further embodiment, the expression of a nucleic acid molecule foran ARM1 protein, for example in combination with a tissue-specificincrease in the activity of a Bax inhibitor-1 protein may take place inthe mesophyll tissue. The reduction of the Armadillo repeat ARM1 proteinquantity in a transgenic plant which for example overexpresses BI-1 inthe mesophyll tissue offers the possibility of generating a complete andcomprehensive fungal resistance in the plant.

In a further embodiment, the increase in the polypeptide quantity,activity or function of a Bax Inhibitor 1 protein from Hordeum vulgare(GenBank Acc. No.: AJ290421), from Nicotiana tabacum (GenBank Acc. No.AF390556), rice (GenBank Acc. No.: AB025926), Arabidopsis (GenBank Acc.No.: AB025927) or tobacco and oilseed rape (GenBank Acc. No.: AF390555,Bolduc N et al. (2003) Planta 216:377-386) or of ROR2 (for example frombarley (GenBank Acc. No.: AY246906), SnAP34 (for example from barley(GenBank Acc. No.: AY247208) and/or of the lumenal binding protein BiPfor example from rice (GenBank Acc. No. AF006825) is effected incombination with the reduction in the protein quantity or activity orfunction of the proteins RacB (for example from barley (GenBank Acc.No.: AJ344223), CSL1 (for example from Arabidopsis (GenBank Acc. No.:NM116593), HvNaOX (for example from barley (GenBank Acc. No.: AJ251717),and/or MLO (for example from barley (GenBank Acc. No. Z83834). As aconsequence, in one embodiment, at least one of the abovementioned geneswhich are suitable for overexpression or increased activity is activatedor overexpressed and/or at least one of the abovementioned genes whichis suitable for reduction is reduced.

An increase in the expression can be obtained as described herein. Anincrease in the expression or function is understood as meaning hereinboth the activation or enhancement of the expression or function of theendogenous protein, including a de novo expression, and an increase orenhancement by expression of a transgenic protein or factor.

For the purposes of the invention, “organism” means “nonhuman organisms”as long as the term relates to a viable multi-celled organism.

For the purposes of the invention, “plants” means all dicotyledonous ormonocotyledonous plants. Preferred are plants which can be subsumedunder the class of the Liliatae (Monocotyledoneae or monocotyledonousplants). The term includes the mature plants, seeds, shoots andseedlings, and parts, propagation material, plant organs, tissue,protoplasts, callus and other cultures, for example cell culturesderived from the above, and all other types of associations of plantcells which give functional or structural units. Mature plants meansplants at any developmental stage beyond the seedling stage. Seedlingmeans a young, immature plant in an early developmental stage.

“Plant” also comprises annual and perennial dicotyledonous ormonocotyledonous plants and includes by way of example, but not bylimitation, those of the genera Bromus, Asparagus, Pennisetum, Lolium,Oryza, Zea, Avena, Hordeum, Secale, Triticum, Sorghum and Saccharum.

In a preferred embodiment, the method is applied to monocotyledonousplants, for example from the family Poaceae, especially preferably tothe genera Oryza, Zea, Avena, Hordeum, Secale, Triticum, Sorghum andSaccharum, very especially preferably to agriculturally important plantssuch as, for example, Hordeum vulgare (barley), Triticum aestivum(wheat), Triticum aestivum subsp. spelta (spelt), Triticale, Avenasativa (oats), Secale cereale (rye), Sorghum bicolor (sorghum), Zea mays(maize), Saccharum officinarum (sugar cane) or Oryza sativa (rice).

“Epidermal tissue” or epidermis means the external tissue layers of theplants. It can be single layered or multiple layered; and there isepidermis-“enriched” gene expression, such as, for example, Cer3, whichcan act as marker, exists; Hannoufa, A. (1996) Plant J. 10 (3), 459-467.

By “epidermis”, the skilled worker preferably means the predominantdermal tissue of primary aerial plant parts, such as of the shoots, theleaves, flowers, fruits and seeds. The epidermal cells excrete awater-repellent layer, the cuticle, towards the outside. The roots aresurrounded by the rhizodermis, which resembles the epidermis in manyways, but also differs substantially therefrom. The epidermis developsfrom the outermost layer of the apical meristem. The origin of therhizodermis, in contrast, is less clear. Phylogenetically speaking, itcan be assigned either to the calyptra or to the primary bark, dependingon the species. A large number of functions can be ascribed to theepidermis: it protects the plant from dehydration and regulates thetranspiration rate. It protects the plant from a wide range of chemicaland physical external factors and against feeding animals and attack byparasites. It is involved in the gas exchange, in the secretion ofcertain metabolites and in the absorption of water. It containsreceptors for light and mechanical stimuli. It therefore acts as signaltransformer between the environment and the plant. In accordance withthe various functions, the epidermis comprises a number of differentlydifferentiated cells. Other aspects are species having specific variantsand different organization of the epidermides in the individual parts ofa plant. Essentially, it consists of three categories of cells: the“actual” epidermal cells, the cells of the stomata and of the trichomes(Greek: trichoma, hair), which are epidermal appendages with differentshapes, structures and functions.

The “actual”, i.e. the least specialized epidermal cells, account formost of the bulk of the cells of the epidermal tissue. In topview, theyappear either polygonal (slab or plate shaped) or elongated. The wallsbetween them are often wavy or sinuate. It is not known what inducesthis shape during development; existing hypotheses only offerunsatisfactory explanations herefor. Elongated epidermal cells can befound in organs or parts of organs that are elongated themselves, thus,for example, in stems, petioles, leaf veins and on the leaves of mostmonocots. The upper surface and undersurface of laminae can be coveredin epidermides with different structures, it being possible for theshape of the cells, the wall thickness and the distribution and numberof specialized cells (stomata and/or trichomes) per unit area to vary. Ahigh degree of variation is also found within individual families, forexample in the Crassulaceae. In most cases, the epidermis consists of asingle layer, though multi-layered water-storing epidermides have beenfound among species from a plurality of families (Moraceae: most Ficusspecies; Piperaceae: Peperonia, Begoniaceae, Malvaceae and the like).Epidermal cells secrete a cuticle to the outside which covers allepidermal surfaces as an uninterrupted film. It may either be smooth orstructured by bulges, rods, folds and furrows. However, the folding ofthe cuticle, which can be observed when viewing the surface, is notalways caused by the formation of cuticular rods. Indeed, there arecases where cuticular folding is merely the expression of the underlyingbulges of the cell wall. Epidermal appendages of various form, structureand function are referred to as trichomes and, in the present context,likewise come under the term “epidermis”. They occur in the form ofprotective hairs, supportive hairs and gland hairs in the form ofscales, different papillae and, in the case of roots, as absorbenthairs. They are formed exclusively by epidermal cells. Frequently, atrichome is formed by only one such cell, however, occasionally, morethan one cell is involved in its formation.

The term “epidermis” likewise comprises papillae. Papillae are bulges ofthe epidermal surface. The textbook example thereof is the papillae onflower surfaces of the pansy (Viola tricolor) and the leaf surfaces ofmany species from tropical rain forests. They impart a velvet-likeconsistency to the surface. Some epidermal cells can form water stores.A typical example is the water vesicles at the surfaces of manyMesembryanthemum species and other succulents. In some plants, forexample in the case of campanula (Campanula persicifolia), the outerwalls of the epidermis are thickened like a lens.

The main biomass of all tissues is the parenchyma. The parenchymatictissues include the mesophyll which, in leaves, can be differentiatedinto palisade parenchyma and spongy parenchyma. Accordingly the skilledworker understands, by mesophyll, a parenchymatic tissue. Parenchymaticcells are always alive, in most cases isodiametric, rarely elongated.The pith of the shoots, the storage tissues of the fruits, seeds, theroot and other underground organs are also to be considered asparenchymas, as is the mesophyll. “Mesophyll tissue” means the foliartissue between the epidermal layers, and consists of palisade tissue,spongy tissue and the vascular bundles of the leaf.

In the leaves of most ferns and phanerogams, especially in the case ofthe dicots and many monocots, the mesophyll is subdivided into palisadeparenchymas and spongy parenchymas. A “typical” leaf is of dorsiventralorganization. In most cases, the palisade parenchyma is at the uppersurface of the leaf immediately underneath the epidermis. The spongyparenchyma fills the underlying space. It is interspersed by avoluminous intercellular system whose gas space is in direct contactwith the external space via the stomata.

The palisade parenchyma consists of elongated cylindrical cells. In somespecies, the cells are irregular, occasionally bifurcate (Y-shaped: armpalisade parenchyma). Such variants are found in ferns, conifers and afew angiosperms (for example in some Ranunculaceae and Caprifoliaceaespecies [example: elder]). Besides the widest-spread organization formwhich has just been described, the following variants have been found:

palisade parenchyma at the leaf undersurface. Particularly conspicuouslyin scaly leaves. (For example arbor vitae (thuja), and in the leaves ofwild garlic (Allium ursinum).

Palisade parenchyma at both leaf surfaces (upper surface andundersurface). Frequently found in plants of dry habitats (xerophytes).Example: prickly lettuce (Lactuca serriola);

Ring-shaped closed palisade parenchyma: in cylindrically organizedleaves and in needles from conifers.

The variability of the cells of the spongy parenchyma, and theorganization of the spongy parenchyma itself, are even more varied thanthat of the palisade parenchyma. It is most frequently referred to asaerenchyma since it comprises a multiplicity of interconnectedintercellular spaces.

The mesophyll may comprise what is known as the assimilation tissue, butthe terms mesophyll and assimilation tissue are not to be usedsynonymously. There are chloroplast-free leaves whose organizationdiffers only to a minor extent from comparable green leaves. As aconsequence, they comprise mesophyll, but assimilation does not takeplace; conversely, assimilation also takes place in, for example,sections of the shoot. Further aids for characterizing epidermis andmesophyll can be found by the skilled worker for example in v.GUTTENBERG, H.: Lehrbuch der Allgemeinen Botanik [Textbook of generalbotany]. Berlin: Akademie-Verlag 1955 (5th Ed.), HABERLANDT, G:Physiologische Pflanzenanatomie [Physiological plant anatomy]. Leipzig:W. Engelmann 1924 (6th Ed.); TROLL, W.: Morphologie der Pflanzen [Plantmorphology]. Volume 1: Vegetationsorgane [Vegetation organs]. Berlin:Gebr. Borntraeger, 1937; TROLL, W.: Praktische Einführung in diePflanzenmorphologie [Practical introduction to plant morphology]. Jena:VEB G. Thieme Verlag 1954/1957; TROLL, W., HÖHN, K.: Allgemeine Botanik[General botany]. Stuttgart: F. Enke Verlag, 1973 (4th Ed.)

As a consequence, epidermis or epidermal cells can be characterized inhistological or biochemical, including molecular-biochemical, terms. Inone embodiment, the epidermis is characterized in biochemical terms. Inone embodiment, the epidermis can be characterized by the activity ofone or more of the following promoters:

WIR5 (=GstA1), acc. X56012, Dudler & Schweizer, unpublished.GLP4, acc. AJ310534; Wei, Y.; (1998) Plant Molecular Biology 36,101-112.GLP2a, acc. AJ237942, Schweizer, P., (1999). Plant J 20, 541-552.Prx7, acc. AJ003141, Kristensen B K, 2001. Molecular Plant Pathology,2(6), 311-317GerA, acc. AF250933; Wu S, 2000. Plant Phys Biochem 38, 685-698OsROC1, acc. AP004656RTBV, acc. AAV62708, MV62707; Klöti, A, 1999, PMB 40, 249-266

Cer3; Hannoufa, A. (1996), Plant J. 10 (3), 459-467.

In another embodiment, the epidermis is characterized in that only someof the promoters are active, for example 2, 3, 5 or 7 or more, but atleast one of the abovementioned promoters is active. In one embodiment,the epidermis is characterized in that all the abovementioned promotersare active in the tissue or the cell.

As a consequence, mesophyll or mesophyll cells can be characterized inbiochemical, including molecular-biological, or histological terms. Inone embodiment, the mesophyll is characterized in biochemical terms. Inone embodiment, the mesophyll can be characterized by the activity ofone or more of the following promoters:

PPCZm1 (=PEPC); Kausch, A. P., (2001) Plant Mot. Biol. 45, 1-15

OsrbcS, Kyozuka et al PlaNT Phys: 1993 102: Kyozuka J, 1993. Plant Phys102, 991-1000

OsPPDK, acc. AC099041.TaGF-2.8, acc. M63223; Schweizer, P., (1999). Plant J 20, 541-552.TaFBPase, acc. X53957.TaWIS1, acc. AF467542; US 200220115849HvBIS1, acc. AF467539; US 200220115849ZmMIS1, acc. AF467514; US 200220115849HvPR1a, acc. X74939; Bryngeisson et al. Molecular Plant-MicrobeInteractions (1994)HvPR1b, acc. X74940; Bryngelsson et al. Molecular Plant-MicrobeInteractions (1994)HvB1,3gluc; acc. AF479647HvPrx8, acc. AJ276227; Kristensen et al MPP 2001 (see above)HvPAL, acc. X97313; Wei, Y.; (1998) Plant Molecular Biology 36, 101-112.

In another embodiment, the mesophyll is characterized in that only someof the promoters are active, for example 2, 3, 5 or 7 or more, but atleast one of the abovementioned promoters is active. In one embodiment,the mesophyll is characterized in that all the abovementioned promotersare active in the tissue or the cell.

In one embodiment, all of the abovementioned promoters are active in theepidermis of a plant which is used or generated in accordance with theinvention or of a plant according to the invention in the epidermis andin the mesophyll. In one embodiment, only some of the abovementionedpromoters are active, for example 2, 5, 7 or more, but at least one ofthe promoters enumerated above is in each case active.

“Nucleic acids” means biopolymers of nucleotides which are linked withone another via phosphodiester bonds (polynucleotides, polynucleicacids). Depending on the type of sugar in the nucleotides (ribose ordeoxyribose), one distinguishes the two classes of the ribonucleic acids(RNA) and the deoxyribonucleic acids (DNA).

The term “crop” means all plant parts obtained by growing plantsagriculturally and collected within the harvesting process.

“Resistance” means the preventing, the repressing, the reducing or theweakening of disease symptoms of a plant as the result of infection by apathogen. The symptoms can be manifold, but preferably comprise thosewhich directly or indirectly lead to an adversely affect on the qualityof the plant, on the quantity of the yield, on the suitability for useas feed or foodstuff, or else which make sowing, growing, harvesting orprocessing of the crop more difficult.

In a preferred embodiment, the following disease symptoms are weakened,reduced or prevented: formation of pustules and hymenia on the surfacesof the affected tissues, maceration of the tissues, spreading necrosesof the tissue, accumulation of mycotoxins, for example from Fusariumgraminearum or F. culmorum, penetration of the epidermis and/or of themesophyll, etc.

“Conferring”, “existing”, “generating” or increasing a pathogenresistance or the like means that the defense mechanisms of a certainplant or in a part of a plant, for example in an organ, a tissue, a cellor an organelle, have an increased resistance to one or more pathogensas the result of using the method according to the invention incomparison with a suitable control, for example the wildtype of theplant (“control plant”, “starting plant”), to which the method accordingto the invention has not been applied, under otherwise identicalconditions (such as, for example, climatic conditions, growingconditions, type of pathogen and the like). Preferably, at least theepidermis and/or mesophyll tissue in a plant, or the organs which havean epidermis and/or mesophyll tissue, have an increased resistance tothe pathogen(s). For example, the resistance in the leaves is increased.In one embodiment, the resistance in lemma, palea and/or glume (antherprimordium) is increased.

In one embodiment, the activity of the protein according to theinvention, Armadillo repeat ARM1, is therefore reduced in theabovementioned organs and tissues.

In this context, the increased resistance preferably manifests itself ina reduced manifestation of the disease symptoms, where diseasesymptoms—in addition to the abovementioned adverse effects—alsocomprises for example the penetration efficiency of a pathogen into theplant or the plant cell, or the proliferation efficiency in or on thesame. Changes in the cell wall structure may, for example, constitute abasic mechanism of pathogen resistance, as shown, for example, in JacobsA K et al. (2003) Plant Cell, 15(11):2503-13.

In the present case, the disease symptoms are preferably reduced by atleast 10% or at least 20%, especially preferably by at least 40% or 60%,very especially preferably by at least 70% or 80%, most preferably by atleast 90% or 95% or more.

For the purposes of the invention, “pathogen” means organisms whoseinteractions with a plant lead to the above-described disease symptoms;in particular, pathogens means organisms from the Kingdom Fungi.Preferably, pathogen is understood as meaning a pathogen whichpenetrates epidermis or mesophyll cells, especially preferably pathogenswhich penetrate plants via stomata and subsequently penetrate mesophyllcells. Organisms which are preferably mentioned in this context arethose from the phyla Ascomycota and Basidiomycota. Especially preferredin this context are the families Blumeriaceae, Pucciniaceae,Mycosphaerellaceae and Hypocreaceae.

Especially preferred are organisms of these families which belong to thegenera Blumeria, Puccinia, Fusarium or Mycosphaerella.

Very especially preferred are the species Blumeria graminis, Pucciniatriticina, Puccinia striiformis, Mycosphaerella graminicola,Stagonospora nodorum, Fusarium graminearum, Fusarium culmorum, Fusariumavenaceum, Fusarium poae and Microdochium nivale.

However, it is to be assumed that the reduction in the expression ofHvARM, its activity or function also brings about a resistance tofurther pathogens.

Especially preferred are Ascomycota such as, for example, Fusariumoxysporum (fusarium wilt on tomato), Septoria nodorurm and Septoriatritici (glume blotch on wheat), Basidiomycetes such as, for example,Puccinia graminis (stem rust on wheat, barley, rye, oats), Pucciniarecondita (leaf rust on wheat), Puccinia dispersa (leaf rust on rye),Puccinia hordei (leaf rust on barley), Puccinia coronata (crown rust onoats).

In preferred embodiments, the method according to the invention leads toa resistance in

-   -   barley to the pathogen:    -   Puccinia graminis f.sp. hordei (barley stem rust), Blumeria        graminis f.sp. hordei (barley mildew, Bgh);    -   in wheat to the pathogens:    -   Fusarium graminearum, Fusarium avenaceum, Fusarium culmorum,        Puccinia graminis f.sp. tritici, Puccinia recondita ftsp.        tritici, Puccinia striiformis, Septoria nodorum, Septoria        tritici, Septoria avenae, Blumeria graminis f.sp. tritici        (barley mildew, Bgt) or Puccinia graminis f.sp. tritici (wheat        stem rust),    -   in maize to the pathogens:    -   Fusarium moniliforme var. subglutinans, Puccinia sorghi or        Puccinia polysora,    -   in sorghum to the pathogens;    -   Puccinia purpurea, Fusarium moniliforme, Fusarium graminearum or        Fusarium oxysporum,    -   in soybean to the pathogens    -   Phakopsora pachyrhizi and Phakopsora meibromae.

“Armadillo repeat Arm1 polypeptide” or “Armadillo repeat ARM1 protein”or “Arm” or “Arm1” and modifications thereof mean in the context of theinvention a protein having one or more Armadillo repeats.

In a particularly preferred embodiment, the invention relates to anArmadillo repeat ARM1 polypeptide which has the activity shown in theexamples. In one embodiment, an Armadillo repeat ARM1 protein isunderstood as meaning a protein with a homology to one of the amino acidsequences shown in SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 32, 34, 36, 38, 42, 44, 60, 61 or 62 or in the figures,for example an Armadillo repeat ARM1 polypeptide from barley (HvARM) asin SEQ ID NO: 2 and/or from rice (Oryza sativa) as shown in SEQ ID NO:4, 6, 8, and/or 10, and/or from tobacco (Nicotiana tabacum) as shown inSEQ ID NO.: 12 and/or from A. thaliana as shown in SEQ ID NO: 14, 16,18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 42, and/or 44, or accordingto one of the consensus sequences as shown in SEQ ID NO.: 60, 61 or 62,or a functional fragment thereof. In one embodiment, the inventionrelates to functional equivalents of the abovementioned polypeptidesequences.

“Polypeptide quantity” means for example the number of molecules, ormoles, of Armadillo repeat ARM1 polypeptide molecules in an organism, atissue, a cell or a cell compartment “Reducing” the polypeptide quantitymeans the molar reduction in the number of Armadillo repeat ARM1polypeptides, in particular of those shown in SEQ ID No: 2, 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 42, 44, 60, 61or 62, in an organism, a tissue, a cell or a cell compartment—forexample by one of the methods described hereinbelow—in comparison with asuitable control, for example the wildtype (control plant) of the samegenus and species to which this method has not been applied, underotherwise identical conditions (such as, for example, cultureconditions, age of the plants and the like). The reduction in thiscontext amounts to at least 5%, preferably at least 10% or at least 20%,especially preferably at least 40% or 60%, very especially preferably atleast 70% or 80%, most preferably at least 90%, 95% or 99% and inparticular 100%.

The present invention furthermore relates to the generation of apathogen resistance by reducing the function or activity of an Armadillorepeat ARM1 polypeptide comprising the sequences shown in SEQ ID No: 2,4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 42,44, 60, 61 or 62 or of a homolog thereof and/or a polypeptide which hasa homology of at least 40% with the above, and/or of a functionalequivalent of the abovementioned polypeptides.

Homology between two nucleic acid sequences is understood as meaning theidentity of the nucleic acid sequence over in each case the entiresequence length, which is calculated by comparison with the aid of theprogram algorithm GAP (Wisconsin Package Version 10.0, University ofWisconsin, Genetics Computer Group (GCG), Madison, USA; Altschul et al.(1997) Nucleic Acids Res. 25:3389ff), setting the following parameters:

Gap weight: 50 Length weight: 3 Average match: 10 Average mismatch: 0

For example, a sequence which has at least 80% homology with thesequence SEQ ID NO: 1 at the nucleic acid level is understood as meaninga sequence which, upon comparison with the sequence SEQ ID NO: 1 by theabove program algorithm with the above parameter set, has at least 80%homology.

Homology between two polypeptides is understood as meaning the identityof the amino acid sequence over the indicated entire sequence lengthwhich is calculated by comparison with the aid of the program algorithmGAP (Wisconsin Package Version 10.0, University of Wisconsin, GeneticsComputer Group (GCG), Madison, USA), setting the following parameters:

Gap weight: 8 Length weight: 2 Average match: 2.912 Average mismatch:−2.003

For example, a sequence which has at least 80% homology at thepolypeptide level with the sequence SEQ ID NO: 2 is understood asmeaning a sequence which, upon comparison with the sequence SEQ ID NO: 2by the above program algorithm with the above parameter set has at least80% homology.

In a preferred embodiment of the present invention, the Armadillo repeatARM1 protein activity, function or polypeptide quantity is reduced inthe plant or in a part of the plant, for example in a plant organ, planttissue, a plant cell or a part of a plant cell, for example aplant-specific organelle.

In one embodiment of the method of the invention, the activity of apolypeptide comprising at least one, preferably two or more, Armadillorepeats is reduced.

In one embodiment, the polypeptide which is reduced in a plant or in apart of the plant does not have a U box in the 5′-UTR.

“Armadillo repeat” means a sequence which comprises the copies arrangedin tandem of a degenerated sequence of approx. 42 amino acids, whichsequence encodes a three-dimensional structure for mediatingprotein-protein interactions (Azevedo et al. (2001) Trends Plant Sci. 6,354-358). For example, the polypeptide employed in the method of theinvention or the polypeptide of the invention has an activity which isinvolved in intracellular signal transduction or in regulating geneexpression within the framework of cellular development of processes.

For example, the Armadillo repeat ARM1 protein is encoded by a nucleicacid molecule comprising a nucleic acid molecule selected from the groupconsisting of:

-   a) nucleic acid molecule which codes for a polypeptide which    comprises the sequence shown in SEQ ID No: 2, 4, 6, 8, 10, 12, 14,    16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 42, 44, 60, 61 or    62;-   b) nucleic acid molecule which comprises at least one polynucleotide    of the sequence according to SEQ ID No: 1, 3, 5, 7, 9, 11, 13, 15,    17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, or 43;-   c) nucleic acid molecule which codes for a polypeptide whose    sequence has 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or    more identity to the sequences SEQ ID No: 2, 4, 6, 8, 10, 12, 14,    16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 42, 44, 60, 61 or    62;-   d) nucleic acid molecule according to (a) to (c) which codes for a    functional fragment or an epitope of the sequences as shown in SEQ    ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,    34, 36, 38, 42, 44, 60, 61 or 62;-   e) nucleic acid molecule which codes for a polypeptide which is    recognized by a monoclonal antibody directed against a polypeptide    which is encoded by the nucleic acid molecules as shown in (a) to    (c); and-   f) nucleic acid molecule which hybridizes under stringent conditions    with a nucleic acid molecule as shown in (a) to (c); or their    part-fragments of at least 15 nucleotides (nt), preferably 20 nt, 30    nt, 50 nt, 100 nt, 200 nt or 500 nt;-   g) nucleic acid molecule which can be isolated from a DNA library    using a nucleic acid molecule as shown in (a) to (c) or their    part-fragments of at least 15 nt, preferably 20 nt, 30 nt, 50 nt,    100 nt, 200 nt or 500 nt, as probe under stringent hybridization    conditions;    or comprises a complementary sequence thereof or constitutes a    functional equivalent thereof.

According to the invention, the activity of the abovementionedpolypeptides is reduced in a plant or a part of a plant, preferably inthe epidermal and/or mesophyll cells of a plant as detailed above.

In one embodiment, the activity of ARM1 is reduced in lemma, paleaand/or glume.

“Epitope” is understood as meaning the regions of an antigen whichdetermine the specificity of the antibodies (the antigenic determinant).

Accordingly, an epitope is the portion of an antigen which actuallycomes into contact with the antibody.

Such antigenic determinants are those regions of an antigen to which theT-cell receptors react and, as a consequence, produce antibodies whichspecifically bind the antigenic determinant/the epitope of an antigen.Accordingly, antigens, or their epitopes, are capable of inducing theimmune response of an organism with the consequence of the formation ofspecific antibodies which are directed against the epitope. Epitopesconsist for example of linear sequences of amino acids in the primarystructure of proteins, or of complex secondary or tertiary proteinstructures. A hapten is understood as meaning a epitope which isdissociated from the context of the antigen environment. Althoughhaptens have by definition an antibody directed against them, haptensare, under certain circumstances, not capable of inducing an immuneresponse in an organism, for example after an injection. To this end,haptens are coupled with carrier molecules. An example which may bementioned is dinitrophenol (DNP), which, after coupling to BSA (bovineserum albumin), has been used for generating antibodies which aredirected against DNP. (Bohn, A., König, W. 1982). Haptens are thereforein particular (frequently low molecular weight or small) substanceswhich, while they themselves do not trigger immune response, will indeedtrigger such a response when coupled to a large molecular carrier.

The antibodies generated thus also include those which can bind to thehapten as such.

In one embodiment, the present invention relates to an antibody againsta polypeptide characterized herein, in particular to a monoclonalantibody which binds a polypeptide which comprises an AA sequence orconsists thereof, as shown in the sequences shown in SEQ ID No: 2, 4, 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42,44, 60, 61 or 62.

Antibodies within the scope of the present invention can be used foridentifying and isolating polypeptides disclosed in accordance with theinvention from organisms, preferably plants, especially preferablymonocotyledonous plants. The antibodies can either be monoclonal,polyclonal or else synthetic in nature or else consist of antibodyfragments such as Fab, Fv or scFv fragments, which are formed byproteolytic degradation. “Single chain” Fv (scFv) fragments aresingle-chain fragments which, linked via a flexible linker sequence onlycomprise the variable regions of the heavy and light antibody chains.Such scFv fragments can also be produced as recombinant antibodyderivatives. A presentation of such antibody fragments on the surface offilamentous phages makes possible the direct selection, from combinatoryphage libraries, of scFv molecules which bind with high affinity.

Monoclonal antibodies can be obtained in accordance with the methoddescribed by Köhler and Milstein (Nature 256 (1975), 495).

“Functional equivalents” of an Armadillo repeat ARM1 protein preferablymeans those polypeptides which have at least 40% homology with thepolypeptides described by the sequences SEQ ID No: 2, 4, 6, 8, 10, 12,14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 42, 44, 60, 61 or 62and which have essentially the same properties or function. Preferably,the homology amounts to 50%, 60%, 70%, 80%, 90%, particularly preferably95%, 97%, 98%, 99% or more.

The functional equivalence can be determined for example by comparingthe phenotypes of test organisms after expression of the polypeptides inquestion, under the most identical conditions possible, or afterreduction of the expression or activity of the polypeptides to becompared, in the source organisms in question.

“Essentially identical properties” of a functional equivalent meansabove all imparting a pathogen-resistant phenotype or imparting orincreasing the pathogen resistance to at least one pathogen whenreducing the polypeptide quantity, activity or function of saidfunctional Armadillo repeat ARM1 protein equivalent in a plant, organ,tissue, part or cells, in particular in epidermal or mesophyll cells ofsame, preferably measured by the penetration efficiency of a pathogen,as shown in the examples.

“Analogous conditions” means that all basic conditions such as, forexample, culture or growth conditions, assay conditions (such asbuffers, temperature, substrates, pathogen concentration and the like)between the experiments to be compared are essentially kept identicaland that the set-ups only differ by the sequence of the Armadillo repeatARM1 polypeptides to be compared, by their source organism and, ifappropriate, by the pathogen.

“Functional equivalents” also means natural or artificial mutationvariants of the Armadillo repeat ARM1 polypeptides as shown in SEQ IDNo: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36,38, 42, 44, 60, 61 or 62 and homologous polypeptides from othermonocotyledonous and dicotyledonous plants which furthermore haveessentially identical properties. Preferred are homologous polypeptidesfrom preferred plants described herein. The sequences from other plants,which sequences are homologous to the Armadillo repeat ARM1 proteinsequences disclosed within the scope of the present invention, can befound readily for example by database search or by screening genelibraries using the Armadillo repeat ARM1 protein sequences as searchsequence or probe.

Functional equivalents can also be derived for example from one of thepolypeptides according to the invention as shown in SEQ ID No: 2, 4, 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 42, 44,60, 61 or 62 by substitution, insertion or deletion and can have atleast 40%, 50%, 60%, preferably at least 80%, by preference at least90%, especially preferably at least 95%, very especially preferably atleast 98% homology with these polypeptides and are distinguished byessentially identical functional properties to the polypeptides as shownin SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,32, 34, 36, 38, 42, 44, 60, 61 or 62.

Functional equivalents are also any nucleic acid molecules which arederived from the nucleic acid sequences according to the invention asshown in SEQ ID No: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27,29, 31, 33, 35, 37, 39, 41, or 43 by substitution, insertion or deletionand have at least 40%, 50%, 60%, preferably 80%, by preference at least90%, especially preferably at least 95%, very especially preferably atleast 98% homology with one of the polynucleotides according to theinvention as shown in SEQ ID No: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,23, 25, 27, 29, 31, 33, 35, 37, 39, 41, or 43 and code for polypeptideswith essentially identical functional properties to polypeptides asshown in SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,30, 32, 34, 36, 38, 42, 44, 60, 61 or 62.

Examples of the functional equivalents of the Armadillo repeat ARM1proteins as shown in SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 32, 34, 36, 38, 42, 44, 60, 61 or 62 which are to bereduced in the method according to the invention can be found byhomology comparisons from databases, from organisms whose genomicsequence is known.

Screening cDNA libraries or genomic libraries of other organisms,preferably of the plant species mentioned further below, which aresuitable as transformation hosts, using the nucleic acid sequencedescribed in SEQ ID No: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, or 43 or parts of the same as probe isalso a method known to the skilled worker for identifying homologs inother species. In this context, the probes derived from the nucleic acidsequence as shown in SEQ ID No: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,23, 25, 27, 29, 31, 33, 35, 37, 39, 41, or 43 have a length of at least20 bp, preferably at least 50 bp, especially preferably at least 100 bp,very especially preferably at least 200 bp, most preferably at least 400bp. The probe can also be one or more kilobases in length, for example 1kb, 1.5 kb or 3 kb. A DNA strand which is complementary to the sequencesdescribed in SEQ ID No: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, or 43, or a fragment of same strand witha length of between 20 bp and several kilobases may also be employed forscreening the libraries.

In the method according to the invention, those DNA molecules whichhybridize under standard conditions with the nucleic acid moleculesdescribed by SEQ ID No: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, or 43 and which code for Armadillorepeat ARM1 proteins, with the nucleic acid molecules which arecomplementary to the above or with parts of the above and which, ascomplete sequences, code for polypeptides which have essentiallyidentical properties, preferably functional properties, to thepolypeptides described in SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 36, 38, 42, 44, 60, 61 or 62, may also beused.

“Standard hybridization conditions” is to be understood in the broadsense and means, depending on the application, stringent or else lessstringent hybridization conditions. Such hybridization conditions aredescribed, inter alia, in Sambrook J, Fritsch E F, Maniatis T et al., inMolecular Cloning (A Laboratory Manual), 2nd edition, Cold Spring HarborLaboratory Press, 1989, pages 9.31-9.57) or in Current Protocols inMolecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.

The skilled worker would choose hybridization conditions from hisspecialist knowledge which allow him to differentiate between specificand unspecific hybridizations.

For example, the conditions during the wash step can be selected fromamong low-stringency conditions (with approximately 2×SSC at 50° C.) andhigh-stringency conditions (with approximately 0.2×SSC at 50° C.,preferably at 65° C.) (20×SSC: 0.3M sodium citrate, 3M NaCl, pH 7.0).Moreover, the temperature during the wash step can be raised fromlow-stringency conditions at room temperature, approximately 22° C., tohigher-stringency conditions at approximately 65° C. The two parameters,salt concentration and temperature can be varied simultaneously or elsesingly, keeping in each case the other parameter constant. During thehybridization, it is also possible to employ denaturant agents such as,for example, formamide or SOS. In the presence of 50% formamide, thehybridization is preferably carried out at 42° C. Some preferredconditions for hybridization and wash step are detailed hereinbelow:

-   (1) Hybridization conditions can be selected for example among the    following conditions:    -   a) 4×SSC at 65° C.,    -   b) 6×SSC at 45° C.,    -   c) 6×SSC, 100 μg/ml denatured fragmented fish sperm DNA at 68°        C.,    -   d) 6×SSC, 0.5% SDS, 100 μg/ml denatured salmon sperm DNA at 68°        C.,    -   e) 6×SSC, 0.5% SDS, 100 μg/ml denatured fragmented salmon sperm        DNA, 50% formamide at 42° C.,    -   f) 50% formamide, 4×SSC at 42° C., or    -   g) 50% (vol/vol) formamide, 0.1% bovine serum albumin, 0.1%        Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer        pH 6.5, 750 mM NaCl, 75 mM sodium citrate at 42° C., or    -   h) 2× or 4×SSC at 50° C. (low-stringency condition),    -   i) 30 to 40% formamide, 2× or 4×SSC at 42° C. (low-stringency        condition),    -   j) 500 mN sodium phosphate buffer pH 7.2, 7% SDS (g/V), 1 mM        EDTA, 10 μg/ml single stranded DNA, 0.5% BSA (g/V) (Church and        Gilbert, Genomic sequencing. Proc. Natl. Acad. Sci. U.S.A.        81:1991. 1984)-   (2) Wash steps can be selected for example among the following    conditions:    -   a) 0.015 M NaCl/0.0015 M sodium citrate/0.1% SDS at 50° C.    -   b) 0.1×SSC at 65° C.    -   c) 0.1×SSC, 0.5% SDS at 68° C.    -   d) 0.1×SSC, 0.5% SDS, 50% formamide at 42° C.    -   e) 0.2×SSC, 0.1% SDS at 42° C.    -   f) 2×SSC at 65° C. (low-stringency condition).

In one embodiment, the hybridization conditions are selected as follows:

A hybridization buffer comprising formamide, NaCl and PEG 6000 ischosen. The presence of formamide in the hybridization bufferdestabilizes double-strand nucleic acid molecules, whereby thehybridization temperature can be lowered to 42° C. without therebyreducing the stringency. The use of salt in the hybridization bufferincreases the renaturation rate of a duplex DNA, in other words thehybridization efficiency. Although PEG increases the viscosity of thesolution, which has a negative effect on the renaturation rates, thepresence of the polymer in the solution increases the concentration ofthe probe in the remaining medium, which increases the hybridizationrate. The composition of the buffer is:

Hybridization buffer 250 mM sodium phosphate buffer pH 7.2 1 mM EDTA 7%SDS (g/v) 250 mM NaCl 10 μg/ml ssDNA 5% polyethylene glycol (PEG) 600040% formamide

The hybridizations are carried out overnight at 42° C. On the followingmorning, the filters are washed 3× with 2×SSC+0.1% SOS for in each caseapproximately 10 minutes.

In a further preferred embodiment of the present invention, an increasein the resistance in the method according to the invention is achievedby

-   (a) reducing the expression of at least one Armadillo repeat ARM1    protein;-   (b) reducing the stability of at least one Armadillo repeat ARM1    protein or of the mRNA molecules which correspond to this Armadillo    repeat ARM1 protein;-   (c) reducing the activity of at least one Armadillo repeat ARM1    protein;-   (d) reducing the transcription of at least one gene which codes for    Armadillo repeat ARM1 protein by expressing an endogenous or    artificial transcription factor; or-   (e) adding, to the food or to the medium, an exonogous factor which    reduces the Armadillo repeat ARM1 protein activity.

“Gene expression” and “expression” are to be understood as beingsynonymous and mean the realization of the information which is storedin a nucleic acid molecule. Reducing the expression of a gene thereforecomprises the reduction of the polypeptide quantity of the encodedprotein, for example of the Armadillo repeat ARM1 polypeptide or of theArmadillo repeat ARM1 protein function. The reduction of the geneexpression of an Armadillo repeat ARM1 protein gene can be realized inmany different ways, for example by one of the methods listedhereinbelow.

“Reduction”, “reducing” or “to reduce” in the context of an Armadillorepeat ARM1 protein or Armadillo repeat ARM1 protein function is to beinterpreted in the broad sense and comprises the partial or essentiallycomplete inhibition or blockage of the functionality of an Armadillorepeat ARM1 polypeptide in a plant or a part, tissue, organ, cells orseeds derived therefrom, based on different cell-biological mechanisms.

Reducing within the meaning of the invention also comprises a quantitivereduction of an Armadillo repeat ARM1 polypeptide down to an essentiallycomplete absence of the Armadillo repeat ARM1 polypeptide (i.e. lack ofdetectability of Armadillo repeat ARM1 protein function or lack ofimmunological detectability of the Armadillo repeat ARM1 protein). Here,the expression of a certain Armadillo repeat ARM1 polypeptide or theArmadillo repeat ARM1 protein function in a cell or an organism ispreferably reduced by more than 50%, especially preferably by more than80%, very especially preferably by more than 90%, in comparison with asuitable control, i.e. to the wildtype of the same type, for example ofthe same genus, species, variety, cultivar and the like (“controlplants”), to which this method has not been applied, under otherwiseessentially identical conditions (such as, for example, cultureconditions, age of the plants and the like).

In accordance with the invention, there are described various strategiesfor reducing the expression of an Armadillo repeat ARM1 protein or anArmadillo repeat ARM1 protein function. The skilled worker recognizesthat a series of further methods is available for influencing theexpression of an Armadillo repeat ARM1 polypeptide or of the Armadillorepeat ARM1 protein function in the desired manner.

In one embodiment, a reduction in the Armadillo repeat ARM1 proteinfunction is achieved in the method according to the invention byapplying at least one method selected from the group consisting of:

-   a) introducing a nucleic acid molecule coding for ribonucleic acid    molecules suitable for forming double-strand ribonucleic acid    molecules (dsRNA), where the sense strand of the dsRNA molecule has    at least 30% homology with the nucleic acid molecule according to    the invention, for example with one of the nucleic acid molecules as    shown in SEQ ID No: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,    27, 29, 31, 33, 35, 37, 39, 41 or 43, or coding for a consensus    sequence as shown in SEQ ID NO.: 60, 61, or 62, or comprises a    fragment of at least 17 base pairs, which has at least 50% homology    with a nucleic acid molecule according to the invention, for example    as shown in SEQ ID No: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,    25, 27, 29, 31, 33, 35, 37, 39, 41 or 43 or coding for a consensus    sequence as shown in SEQ ID NO.: 60, 61 or 62, or with a functional    equivalent of same, or introducing (an) expression cassette(s) which    ensure(s) their expression.-   b) introducing a nucleic acid molecule coding for an antisense    ribonucleic acid molecule which has at least 30% homology with the    noncoding strand of one of the nucleic acid molecules according to    the invention, for example a nucleic acid molecule as shown in SEQ    ID No: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,    33, 35, 37, 39, 41 or 43 or coding for a consensus sequence as shown    in SEQ ID NO.: 60, 61 or 62, or comprising a fragment of at least 15    base pairs with at least 50% homology with a noncoding strand of a    nucleic acid molecule according to the invention, for example as    shown in SEQ ID No: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,    27, 29, 31, 33, 35, 37, 39, 41 or 43 or coding for a consensus    sequence as shown in SEQ ID NO.: 60, 61 or 62, or with a functional    equivalent thereof. Comprised are those methods in which the    antisense nucleic acid sequence against an Armadillo repeat ARM1    protein gene (i.e. genomic DNA sequences) or an Armadillo repeat    ARM1 protein gene transcript (i.e. RNA sequences). Also comprised    are α-anomeric nucleic acid sequences.-   c) introducing a ribozyme which specifically cleaves, for example    catalytically, the ribonucleic acid molecules encoded by a nucleic    acid molecule according to the invention, for example as shown in    SEQ ID No: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29,    31, 33, 35, 37, 39, 41 or 43 or coding for a consensus sequence as    shown in SEQ ID NO.: 60, 61 or 62 or by their functional    equivalents, by introducing an expression cassette which ensures the    expression of such a ribozyme.-   d) introducing an antisense nucleic acid molecule as specified in    b), in combination with a ribozyme or with an expression cassette    which ensures the expression of the ribozyme.-   e) introducing nucleic acid molecules coding for sense ribonucleic    acid molecules of a polypeptide according to the invention, for    example as shown in the sequences SEQ ID No: 2, 4, 6, 8, 10, 12, 14,    16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 42, 44, 60, 61 or    62, for polypeptides with at least 40% homology with the amino acid    sequence of a protein according to the invention, or is a functional    equivalent thereof.-   f) introducing a nucleic acid sequence coding for a    dominant-negative polypeptide suitable for suppressing the Armadillo    repeat ARM1 protein function, or introducing an expression cassette    which ensures the expression of this nucleic acid sequence.-   g) introducing a factor which can specifically bind Armadillo repeat    ARM1 polypeptides or the DNA or RNA molecules coding for these    polypeptides, or introducing an expression cassette which ensures    the expression of this factor.-   h) introducing a viral nucleic acid molecule which brings about a    degradation of mRNA molecules which code for Armadillo repeat ARM1    protein, or introducing an expression cassette which ensures the    expression of this nucleic acid molecule.-   i) introducing a nucleic acid construct suitable for inducing a    homologous recombination on genes coding for Armadillo repeat ARM1    protein.-   j) introducing one or more mutations into one or more coding gene(s)    coding for Armadillo repeat ARM1 proteins for generating a loss of    function (for example generation of stop codons, reading-frame    shifts and the like).

Each one of these methods can bring about a reduction in the Armadillorepeat ARM1 protein expression or Armadillo repeat ARM1 protein functionfor the purposes of the invention. A combined use is also feasible.Further methods are known to the skilled worker and can comprise thehindering or prevention of the processing of the Armadillo repeat ARM1polypeptide, of the transport of the Armadillo repeat ARM1 polypeptideor its mRNA, inhibition of the ribosome attachment, inhibition of theRNA splicing, induction of an Armadillo repeatARM1-protein-RNA-degrading enzyme and/or inhibition of the translationalelongation or termination.

A reduction in the Armadillo repeat ARM1 protein function or Armadillorepeat ARM1 polypeptide quantity is preferably achieved by a reducedexpression of an endogenous Armadillo repeat ARM1 protein gene.

The individual preferred processes are described briefly hereinbelow:

a) Introducing a Double-Stranded Armadillo Repeat ARM1 Protein RNANucleic Acid Sequence (Armadillo Repeat ARM1 Protein dsRNA)

The method of regulating genes by means of double-stranded RNA(“double-stranded RNA interference”; dsRNAi) has been described manytimes for animal and plant organisms (e.g. Matzke M A et al. (2000)Plant Mol Biol 43:401-415; Fire A. et al (1998) Nature 391:806-811; WO99/32619; WO 99/53050; WO 00/68374; WO 00/44914; WO 00/44895; WO00/49035; WO 00/63364). Efficient gene suppression can also bedemonstrated in the case of transient expression, or following thetransient transformation, for example as the result of a biolistictransformation (Schweizer P et al. (2000) Plant J 2000 24: 895-903).dsRNAi processes are based on the phenomenon that simultaneouslyintroducing the complementary strand and counterstrand of a genetranscript suppresses the expression of the corresponding gene in ahighly efficient manner. The phenotype caused is very similar to that ofa corresponding knock-out mutant (Waterhouse P M et al. (1998) Proc NatlAcad Sci USA 95:13959-64).

The dsRNAi method has proved to be particularly efficient andadvantageous when reducing the protein expression (WO 99/32619).

With regard to the double-stranded RNA molecules, Armadillo repeat ARM1protein nucleic acid sequence preferably means one of the sequences asshown in SEQ ID No: 1, 3, 5, 7, 9, 11; 13, 15, 17, 19, 21, 23, 25, 27,29, 31, 33, 35, 37, 39, 41 or 43, or coding for a consensus sequence asshown in SEQ ID NO.: 60, 61 or 62, or sequences which are essentiallyidentical to those, preferably which have at least 50%, 60%, 70%, 80% or90% or more identity to these, for example approximately 95%, 96%, 97%,98%, 99% or more identity to these, or fragments of these with a lengthof at least 17 base pairs. “Essentially identical” means here that thedsRNA sequence may also have insertions, deletions and individual pointmutations in comparison with the Armadillo repeat ARM1 protein targetsequence while still bringing about an efficient reduction in theexpression. In one embodiment, the homology as defined above is at least50%, for example approximately 80%, or approximately 90%, orapproximately 100%, between the “sense” strand of an inhibitory dsRNAand a subsection of an Armadillo repeat ARM1 protein nucleic acidsequence (or between the “antisense” strand and the complementary strandof an Armadillo repeat ARM1 protein nucleic acid sequence). The lengthof the subsection is approximately 17 bases or more, for exampleapproximately 25 bases, or approximately 50 bases, approximately 100bases, approximately 200 bases or approximately 300 bases.Alternatively, an “essentially identical” dsRNA can also be defined as anucleic acid sequence which is capable of hybridizing under stringentconditions with a part of an Armadillo repeat ARM1 protein genetranscript.

The “antisense” RNA strand, too, can have insertions, deletions andindividual point mutations in comparison with the complement of the“sense” RNA strand. The homology is preferably at least 80%, for exampleapproximately 90%, or approximately 95%, or approximately 100%, betweenthe “antisense” RNA strand and the complement of the “sense” RNA strand.

“Subsection of the “sense” RNA transcript” of a nucleic acid moleculecoding for an Armadillo repeat ARM1 polypeptide or a functionalequivalent thereof means fragments of an RNA or mRNA transcribed by anucleic acid molecule coding for an Armadillo repeat ARM1 polypeptide ora functional equivalent thereof, preferably by an Armadillo repeat ARM1protein gene. In this context, the fragments preferably have a sequencelength of approximately 20 bases or more, for example approximately 50bases, or approximately 100 bases, or approximately 200 bases, orapproximately 500 bases. Also comprised is the complete transcribed RNAor mRNA.

The dsRNA can consist of one or more strands of polymerizedribonucleotides. Modifications both of the sugar-phosphate backbone andof the nucleosides may also be present. For example, the phosphodiesterbonds of the natural RNA can be modified in such a way that theycomprise at least one nitrogen or sulfur heteroatom.

Bases can be modified in such a way that the activity of, for example,adenosin deaminase is restricted. Such and further modifications aredescribed hereinbelow in the methods of stabilizing antisense RNA.

To achieve the same purpose, it is, of course, also possible tointroduce, into the cell or the organism, a plurality of individualdsRNA molecules, each of which comprises one of the above-definedribonucleotide sequence segments.

The dsRNA can be prepared enzymatically or fully or partially bychemical synthesis.

If the two strands of the dsRNA are to be combined in one cell or plant,this can be accomplished in various ways,

-   a) transformation of the cell or plant with a vector which comprises    both expression cassettes,-   b) cotransformation of the cell or plant with two vectors, where one    comprises the expression cassettes with the “sense” strand while the    other one comprises the expression cassettes with the “antisense”    strand, and/or-   c) hybridization of two plants which have been transformed with in    each case one vector, where one comprises the expression cassettes    with the “sense” strand, while the other one comprises the    expression cassettes with the “antisense” strand.

The formation of the RNA duplex can be initiated either externally orinternally of the cell. As described in WO 99/53050, the dsRNA can alsocomprise a hairpin structure, by linking “sense” and “antisense” strandby means of a “linker” (for example an intron). The autocomplementarydsRNA structures are preferred since they only require the expression ofa construct and always comprise the complementary strands in anequimolar ratio.

The expression cassettes coding for the “antisense” or “sense” strand ofa dsRNA or for the autocomplementary strand of the dsRNA are preferablyinserted into a vector and stably (for example using selection markers)inserted into the genome of a plant using the methods describedhereinbelow in order to ensure permanent expression of the dsRNA.

The dsRNA can be introduced using a quantity which makes possible atleast one copy per cell. Higher quantities (for example at least 5, 10,100, 500 or 1000 copies per cell) can make, if appropriate, a moreefficient reduction.

In order to bring about an efficient reduction in the Armadillo repeatARM1 protein expression, 100% sequence identity between dsRNA and anArmadillo repeat ARM1 protein gene transcript or the gene transcript ofa functionally equivalent gene is a possible embodiment, but notnecessarily required. Accordingly, there is the advantage that themethod tolerates sequence deviations as they can exist as the result ofgenetic mutations, polymorphisms or evolutionary divergences. The largenumber of highly conserved amino acid residues between differentArmadillo repeat ARM1 protein sequences of different plants, as shown inthe figures with reference to the consensus sequences, allows theconclusion that this polypeptide is highly conserved within plants, sothat the expression of a dsRNA derived from one of the disclosedArmadillo repeat ARM1 protein sequences as shown in SEQ ID No: 1, 3, 5,7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or43 will also have an advantageous effect in other plant species.

As the result of the high number of conserved residues and of thehomology between the individual Armadillo repeat ARM1 polypeptides andtheir functional equivalents, it may also be possible to suppress theexpression of further homologous Armadillo repeat ARM1 polypeptidesand/or their functional equivalents of the same organism, or else theexpression of Armadillo repeat ARM1 polypeptides in other, relatedspecies, using a single dsRNA sequence which has been generated startingfrom a specific Armadillo repeat ARM1 protein sequence of an organism.For this purpose, the dsRNA preferably comprises sequence regions ofArmadillo repeat ARM1 protein gene transcripts which correspond toconserved regions. Said conserved regions can be derived readily fromsequence alignments, for example as shown in the figures. It ispreferred to derive dsRNA sequences from the conserved regions of theconsensus sequence which are shown in the figures. Regions which areregarded as being particularly conserved are: AA702 to AA739, AA742 toAA752, AA760 to AA762, AA771 to 779, AA789 to AA790, AA799 to AA821,AA829 to AA843, M879 to AA905, AA924 to AA939, of the consensus sequencedepicted in the figures.

A dsRNA can be synthesized chemically or enzymatically. To this end, itis possible to use cellular RNA polymerases or bacteriophage RNApolymerases (such as, for example, T3, T7 or SP6 RNA polymerase).Suitable methods for the in vitro expression of RNA are described (WO97/32016; U.S. Pat. No. 5,593,874; U.S. Pat. No. 5,698,425, U.S. Pat.No. 5,712,135, U.S. Pat. No. 5,789,214, U.S. Pat. No. 5,804,693). AdsRNA which has been synthesized chemically or enzymatically in vitrocan be purified from the reaction mixture fully or in part, for exampleby extraction, precipitation, electrophoresis, chromatography orcombinations of these methods, before it is introduced into a cell,tissue or organism. The dsRNA can be introduced into the cell directlyor else applied extracellularly (for example into the interstitialspace).

However, it is preferred to transform the plant stably with anexpression construct which realizes the expression of the dsRNA.Suitable methods are described hereinbelow.

b) Introduction of an Armadillo Repeat ARM1 Protein Antisense NucleicAcid Sequence

Methods of suppressing a certain polypeptide by preventing theaccumulation of its mRNA by means of the “antisense” technology havebeen described many times, including in plants (Sheehy et al. (1988)Proc Natl Acad Sci USA 85; 8805-8809; U.S. Pat. No. 4,801,340; Mol J Net al. (1990) FEBS Lett 268(2):427-430). The antisense nucleic acidmolecule hybridizes with, or binds to, the cellular mRNA and/or genomicDNA coding for the callose synthase target polypeptide to be suppressed.The transcription and/or translation of the target polypeptide isthereby suppressed. The hybridization can be accomplished in atraditional manner via the formation of a stable duplex or, in the caseof genomic DNA, by binding the antisense nucleic acid molecule to theduplex of the genomic DNA as the result of specific interaction in thelarge groove of the DNA helix.

An antisense nucleic acid molecule suitable for reducing an Armadillorepeat ARM1 polypeptide can be derived using the nucleic acid sequencewhich codes for this polypeptide, for example the nucleic acid moleculeaccording to the invention as shown in SEQ ID No: 1, 3, 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 43 or anucleic acid molecule coding for a functional equivalent thereoffollowing Watson's and Crick's base-pairing rules. The antisense nucleicacid molecule can be complementary to all of the transcribed mRNA of thesaid polypeptide, be limited to the coding region or else only consistof an oligonucleotide which is complementary to part of the coding ornoncoding sequence of the mRNA. Thus, for example, the oligonucleotidecan be complementary to the region which comprises the translation startfor said polypeptide. Antisense nucleic acid molecules can have a lengthof, for example, 20, 25, 30, 35, 40, 45 or 50 nucleotides, but they mayalso be longer and comprise 100, 200, 500, 1000, 2000 or 5000nucleotides. Antisense nucleic acid molecules can be expressedrecombinantly or synthesized chemically or enzymatically, using methodsknown to the skilled worker. In the case of chemical synthesis, naturalor modified nucleotides can be used. Modified nucleotides can impart anincreased biochemical stability to the antisense nucleic acid moleculeand lead to an increased physical stability of the duplex formed ofantisense nucleic acid sequence and sense target sequence. Exampleswhich can be used are phosphorus thioate derivatives andacridine-substituted nucleotides such as 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)-uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylqueosine,inosine, N6-isopentenyladenine, 1-methyl-guanine, 1-methylinosine,2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine,5-methylcytosine, N6-adenine, 7-methylguanine,5-methylamino-methyluracil, 5-methoxyaminomethyl-2-thiouracil,β-D-mannosylqueosine, 5′-methoxy-carboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid,pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil,2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acidmethyl ester, uracil-5-oxyacetic acid, 5-methyl-2-thiouracil,3-(3-amino-3-N2-carboxypropyl)uracil and 2,6-diaminopurine.

In a further preferred embodiment, the expression of an Armadillo repeatARM1 polypeptide can be inhibited by nucleic acid molecules which arecomplementary to a conserved region (for example a region which has beenconserved as described above) or to a regulatory region of an Armadillorepeat ARM1 protein gene (for example an Armadillo repeat ARM1 proteinpromoter and/or enhancer) and which form triple-helical structures withthe DNA double helix therein, so that the transcription of the Armadillorepeat ARM1 protein gene is reduced. Suitable methods have beendescribed (Helene C (1991) Anticancer Drug Res 6(6):569-84; Helene C etal. (1992) Ann NY Acad Sci 660:27-36; Maher L J (1992) Bioassays14(12):807-815).

In a further embodiment, the antisense nucleic acid molecule can be anα-anomeric nucleic acid. Such α-anomeric nucleic acid molecules formspecific double-stranded hybrids with complementary RNA in which—asopposed to the conventional β-nucleic acids—the two strands run inparallel with one another (Gautier C et al. (1987) Nucleic Acids Res15:6625-6641). The antisense nucleic acid molecule can furthermore alsocomprise 2′-O-methylribonucleotides (Inoue et al. (1987) Nucleic AcidsRes 15:6131-6148) or chimeric RNA-DNA analogs (Inoue et al. (1987) FEBSLett 215:327-330).

c) Introduction of a Ribozyme which Specifically, for ExampleCatalytically, Cleaves the Ribonucleic Acid Molecules Coding forArmadillo Repeat Protein.

Catalytic RNA molecules or ribozymes can be adapted to any target RNAand cleave the phosphodiester backbone at specific positions, wherebythe target RNA is functionally deactivated (Tanner N K (1999) FEMSMicrobiol Rev 23(3):257-275). As the result, the ribozyme is notmodified itself, but is capable of cleaving further target RNA moleculesin an analogous manner, whereby it obtains the characteristics of anenzyme.

In this manner, it is possible to use ribozymes (for example hammerheadribozymes; Haselhoff and Gerlach (1988) Nature 334:585-591) in order tocleave the mRNA of an enzyme to be suppressed, for example callosesyntheses, and to prevent translation. Methods of expressing ribozymesfor reducing certain polypeptides are described in (EP 0 291 533, EP 0321 201, EP 0 360 257). A ribozyme expression has also been described inplant cells (Steinecke P et al. (1992) EMBO J 11(4):1525-1530; de FeyterR et al. (1996) Mol Gen Genet. 250(3):329-338). Ribozymes can beidentified from a library of various ribozymes via a selection process(Bartel D and Szostak J W (1993) Science 261:1411-1418). Preferably, thebinding regions of the ribozyme hybridize with the conserved regions ofthe ARM protein as described above.

d) Introduction of an Armadillo Repeat ARM1 Protein Antisense NucleicAcid Sequence in Combination with a Ribozyme.

The above-described antisense strategy can advantageously be coupledwith a ribozyme method. The incorporation of ribozyme sequences into“antisense” RNAs imparts this enzyme-like, RNA-cleaving characteristicto precisely these antisense RNAs and thus increases their efficiency inthe inactivation of the target RNA. The preparation and use of suitableribozyme “antisense” RNA molecules is described, for example, inHaselhoff et al. (1988) Nature 334: 585-591.

The ribozyme technology can increase the efficiency of an antisensestrategy. Suitable target sequences and ribozymes can be determined forexample as described in “Steinecke P, Ribozymes, Methods in Cell Biology50, Galbraith et al. eds., Academic Press, Inc. (1995), p. 449-460”, bycalculating the secondary structure of ribozyme RNA and target RNA andby their interaction (Bayley C C et al. (1992) Plant Mol Biol.18(2):353-361; Lloyd A M and Davis R W et al. (1994) Mol Gen Genet.242(6):653-657). For example, it is possible to construct derivatives ofthe Tetrahymena L-19 IVS RNA which derivatives have complementaryregions to the mRNA of the Armadillo repeat ARM1 protein to besuppressed (see also U.S. Pat. No. 4,987,071 and U.S. Pat. No.5,116,742).

e) Introduction of an Armadillo Repeat ARM1 Protein Sense Nucleic AcidSequence for Inducing a Cosuppression

The expression of an Armadillo repeat ARM1 protein nucleic acid sequencein sense orientation can lead to a cosuppression of the correspondinghomologous, endogenous gene. The expression of sense RNA with homologyto an endogenous gene can reduce or cancel the expression of the former,similar to what has been described for antisense approaches (Jorgensenet al. (1996) Plant Mol Biol 31(5):957-973; Goring et al. (1991) ProcNatl Acad Sci USA 88:1770-1774; Smith et al. (1990) Mol Gen Genet224:447-481; Napoli et al. (1990) Plant Cell 2:279-289; Van der Krol etal. (1990) Plant Cell 2:291-99). Here, the construct introduced canrepresent the homologous gene to be reduced either fully or only inpart. The possibility of translation is not required. The application ofthis technology to plants is described for example in Napoli et al.(1990) The Plant Cell 2: 279-289 and in U.S. Pat. No. 5,034,323.

The cosuppression is preferably realized using a sequence which isessentially identical to at least part of the nucleic acid sequencecoding for an Armadillo repeat ARM1 protein or a functional equivalentthereof, for example of the nucleic acid molecule according to theinvention, for example of the nucleic acid sequence as shown in SEQ IDNo: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41 or 43, or of the nucleic acid sequence coding for afunctional equivalent thereof.

f) Introduction of Nucleic Acid Sequences Coding for a Dominant-NegativeArmadillo Repeat ARM1 Protein.

The activity of an Armadillo repeat ARM1 protein can probably also bereduced by expression of a dominant-negative variant of this Armadillorepeat ARM1 protein. Methods of reducing the function or activity of apolypeptide by means of coexpression of its dominant-negative form areknown to the skilled worker (Lagna G and Hemmati-Brivanlou A (1998)Current Topics in Developmental Biology 36:75-98; Perlmutter R M andAlberola-lla J (1996) Current Opinion in Immunology 8(2):285-90;Sheppard D (1994) American Journal of Respiratory Cell & MolecularBiology, 11(1):1-6; Herskowitz I (1987) Nature 329(6136):219-22).

A dominant-negative Armadillo repeat ARM1 protein variant can beaccomplished for example by altering amino acid residues which are partof the Armadillo repeat ARM1 and, as the result of their mutation, thepolypeptide loses its function. Amino acid residues which are preferablyto be mutated are those which are conserved in the Armadillo repeat ARM1proteins of different organisms. Such conserved regions can bedetermined for example by means of computer-aided comparison(“alignment”). These mutations for obtaining a dominant-negativeArmadillo repeat ARM1 protein variant are preferably carried out at thelevel of the nucleic acid sequence coding for Armadillo repeat ARM1proteins. A suitable mutation can be realized for example byPCR-mediated in vitro mutagenesis using suitable oligonucleotideprimers, by means of which the desired mutation is introduced. Methodswhich are known to the skilled worker are used for this purpose. Forexample, the “LA PCR in vitro Mutagenesis Kit” (Takara Shuzo, Kyoto) canbe used for this purpose.

g) Introduction of Armadillo Repeat ARM1 Protein Genes, RNAs orPolypeptide-Binding Factors.

A reduction of an Armadillo repeat ARM1 protein/gene expression is alsopossible using specific DNA-binding factors, for example using factorsof the zinc finger transcription factor type. These factors attach tothe genomic sequence of the endogenous target gene, preferably in theregulatory regions, and bring about a repression of the endogenous gene.The use of such a method makes possible the reduction of the expressionof an endogenous Armadillo repeat ARM1 protein gene without it beingnecessary to recombinantly manipulate the sequence of the latter.Suitable methods for the preparation of suitable factors are described(Dreier B et al. (2001) J Biol Chem 276(31):29466-78; Dreier B et al.(2000) J Mol Biol 303(4):489-502; Beerli R R et al. (2000) Proc NatlAcad Sci USA 97 (4):1495-1500; Beerli R R et al. (2000) J Biol Chem275(42):32617-32627; Segal D J and Barbas C F 3rd. (2000) Curr Opin ChemBiol 4(1):34-39; Kang J S and Kim J S (2000) J Biol Chem275(12):8742-8748; Beerli R R et al. (1998) Proc Natl Acad Sci USA95(25):14628-14633; Kim J S et al. (1997) Proc Natl Acad Sci USA94(8):3616-3620; Klug A (1999) J Mol Biol 293(2):215-218; Tsai S Y etal. (1998) Adv Drug Deliv Rev 30(1-3):23-31; Mapp A K et al. (2000) ProcNatl Acad Sci USA 97(8):3930-3935; Sharrocks A D et al. (1997) Int JBiochem Cell Biol 29(12):1371-1387; Zhang L et al. (2000) J Biol Chem275(43):33850-33860).

The selection of these factors can be accomplished using a suitableportion of an Armadillo repeat ARM1 protein gene. This segment ispreferably located in the region of the promoter region. However, forthe purpose of suppressing a gene, it may also be located in the regionof the coding exons or introns. The corresponding segments areobtainable for the skilled worker by means of database search from thegene library or, starting from an Armadillo repeat ARM1 protein cDNAwhose gene is not present in the gene library, by screening a genomiclibrary for corresponding genomic clones. The methods required for thispurpose are known to the skilled worker.

Furthermore, it is possible to introduce, into a cell, factors whichthemselves inhibit the Armadillo repeat ARM1 protein target polypeptide.The polypeptide-binding factors can be, for example, aptamers (Famulok Mand Mayer G (1999) Curr Top Microbiol Immunol 243:123-36) or antibodiesor antibody fragments. The preparation of these factors is described andknown to the skilled worker. For example, a cytoplasmic scFv antibodyhas been employed for modulating the activity of the phytochrome Aprotein in recombinantly modified tobacco plants (Owen M et al. (1992)Biotechnology (NY) 10(7):790-794; Franken E et al. (1997) Curr OpinBiotechnol 8(4):411-416; Whitelam (1996) Trend Plant Sci 1:286-272).

Gene expression can also be suppressed by customized,low-molecular-weight synthetic compounds, for example of the polyamidetype (Dervan P B and Bürli R W (1999) Current Opinion in ChemicalBiology 3:688-693; Gottesfeld J M et al. (2000) Gene Expr 9(1-2):77-91).These oligomers consist of the units 3-(dimethylamino)-propylamine,N-methyl-3-hydroxypyrrole, N-methylimidazole and N-methylpyrrole and canbe adapted to each segment of double-stranded DNA in such a way thatthey bind into the major group in a sequence-specific fashion and blockthe expression of the gene sequences therein. Suitable methods aredescribed (see, inter alia, Bremer R E et al. (2001) Bioorg Med Chem.9(8):2093-103; Ansari A Z et al. (2001) Chem Biol. 8(6):583-92;Gottesfeld J M et al. (2001) J Mol Biol. 309(3):615-29; Wurtz N R et al.(2001) Org Lett 3(8):1201-3; Wang C C et al. (2001) Bioorg Med Chem9(3):653-7; Urbach A R and Dervan P B (2001) Proc Natl Acad Sci USA98(8):4343-8; Chiang S Y et al. (2000) J Biol Chem. 275(32):24246-54).

h) Introduction of the Viral Nucleic Acid Molecules and ExpressionConstructs which Bring about the Degradation of Armadillo Repeat ARM1Protein RNA.

The Armadillo repeat ARM1 protein expression can also be realizedefficiently by induction of the specific Armadillo repeat ARM1 proteinRNA degradation by the plant with the aid of a viral expression system(Amplikon) (Angell, S M et al. (1999) Plant J. 20(3):357-362). Thesesystems—also referred to as “VIGS” (viral-induced genesilencing)—introduce, by means of viral vectors, nucleic acid sequenceswith homology to the transcripts to be suppressed into the plant.Transcription is then cancelled, probably mediated by plant defensemechanisms against viruses. Suitable techniques and methods aredescribed (Ratcliff F et al. (2001) Plant J 25(2):237-45; Fagard M andVaucheret H (2000) Plant Mol Biol 43(2-3):285-93; Anandalakshmi R et al.(1998) Proc Natl Acad Sci USA 95(22):13079-84; Ruiz M T (1998) PlantCell 10(6): 937-46).

The methods of the dsRNAi, of cosuppression by means of sense RNA and of“VIGS” (“virus-induced gene silencing”) are also referred to as“post-transcriptional gene silencing” (PTGS). PTGS methods areparticularly advantageous because the demands for the homology betweenthe endogenous gene to be suppressed and the recombinantly expressedsense or dsRNA nucleic acid sequence are less stringent than, forexample, in a traditional antisense approach. Suitable homology criteriaare mentioned in the description of the dsRNAI method and can generallybe applied to PTGS methods or dominant-negative approaches. As theresult of the high degree of homology between the Armadillo repeat ARM1proteins from maize, wheat, rice and barley, it can be concluded thatthis polypeptide is highly conserved in plants. Thus, it is probablyalso possible, using the Armadillo repeat ARM1 protein nucleic acidmolecules as they are shown herein, in particular by means of thenucleic acid molecules which are derived from the consensus sequences,or else for example from the nucleic acid molecules from Arabidopsis,barley, maize or rice, also efficiently to suppress the expression ofhomologous Armadillo repeat ARM1 polypeptides in other species withoutthe isolation and structure elucidation of the Armadillo repeat ARM1protein homologs found in these species being compulsory. Thissubstantially simplifies the labor required.

i) Introduction of a Nucleic Acid Construct Suitable for Inducing aHomologous Recombination on Genes Coding for Armadillo Repeat ARM1Proteins, for Example for the Generation of Knockout Mutants.

To generate a homologously-recombinant organism with reduced Armadillorepeat ARM1 protein function, one uses for example a nucleic acidconstruct which comprises at least part of an endogenous Armadillorepeat ARM1 protein gene which is modified by a deletion, addition orsubstitution of at least one nucleotide, for example in the conservedregions, in such a way that the functionality is reduced or entirelynullified.

For example, the primary, secondary, tertiary or quaternary structurecan be disrupted, for example in such a manner that the binding abilityof one or more Armadillo repeats no longer exists. Such a disruption canbe accomplished for example by the mutation of one or more residueswhich are indicated in the consensus sequence as being conserved orhighly conserved.

The modification can also relate to the regulatory elements (for examplethe promoter) of the gene, so that the coding sequence remainsunaltered, but that expression (transcription and/or translation) doesnot take place and is reduced.

In the case of conventional homologous recombination, the modifiedregion is flanked at its 5′ and 3′ terminus by further nucleic acidsequences which must be of sufficient length for making possible therecombination. As a rule, the length is in the range of from severalhundred or more bases up to several kilobases (Thomas K R and Capecchi MR (1987) Cell 51:503; Strepp et al. (1998) Proc Natl Acad Sci USA95(8):4368-4373). To carry out the homologous recombination, the hostorganism—for example a plant—is transformed with the recombinationconstruct using the methods described hereinbelow, and clones which haveundergone successful recombination are selected using for example aresistance to antibiotics or herbicides.

j) Introduction of Mutations into Endogenous Armadillo Repeat ARM1Protein Genes for Generating a Loss of Function (for Example Generationof Stop Codons, Reading-Frame Shifts and the Like)

Further suitable methods for reducing the Armadillo repeat ARM1 proteinfunction are the introduction of nonsense mutations into endogenousArmadillo repeat ARM1 protein genes, for example by means of generationof knockout mutants with the aid of, for example, T-DNA mutagenesis(Koncz et al. (1992) Plant Mol Biol 20(5):963-976), ENU(N-ethyl-N-nitrosourea)-mutagenesis or homologous recombination (Hohn Band Puchta (1999) H Proc Natl Acad Sci USA 96:8321-8323.) or EMSmutagenesis (Birchler J A, Schwartz D. Biochem Genet. 1979 December;17(11-12):1173-80; Hoffmann G R. Mutat Res. 1980 January; 75(1):63-129).Point mutations can also be generated by means of DNA-RNA hybridoligonucleotides, which are also known as “chimeraplasty” (Zhu et al.(2000) Nat Biotechnol 18(5):555-558, Cole-Strauss et al. (1999) NuclAcids Res 27(5):1323-1330; Kmiec (1999) Gene therapy American Scientist87(3):240-247).

The cell- or tissue-specific reduction in the activity of an sARM1 canbe effected for example by expressing a suitable construct, which, forexample, an abovementioned nucleic acid molecule, for example theantisense RNA, dsRNA, RNAi, ribozyme, with a suitable tissue-specificpromoter, for example a promoter as described herein as being specificfor epidermis or mesophyll.

For the purposes of the present invention, “mutations” means themodification of the nucleic acid sequence of a gene variant in a plasmidor in the genome of an organism. Mutations can arise for example as theresult of errors in the replication, or they can be caused by mutagens.While the spontaneous mutation rate in the cell genome of organisms isvery low, the skilled worker is familiar with a multiplicity ofbiological, chemical or physical mutagens.

Mutations comprise substitutions, additions, deletions of one or morenucleic acid residues. Substitutions are understood as meaning theexchange of individual nucleic acid bases; one distinguishes betweentransitions (substitution of a purine base for a purine base, or of apyrimidine base for a pyrimidine base) and transversions (substitutionof a pyrimidine base for a purine base (or vice versa)).

Additions or insertions are understood as meaning the incorporation ofadditional nucleic acid residues into the DNA, it being possible toresult in reading-frame shifts. In the case of such reading-frameshifts, one distinguishes between “in-frame” insertions/additions and“out-of-frame” insertions. In the case of the “in-frame”insertions/additions, the reading frame is retained, and a polypeptidewhich is enlarged by the number of the amino acids encoded by theinserted nucleic acids results. In the case of “out-of-frame”insertions/additions, the original reading frame is lost, and theformation of a complete and functional polypeptide is no longer possiblein many cases, naturally dependent on the location of the mutation.

Deletions describe the loss of one or more base pairs, which likewiselead to “in-frame” or “out-of-frame” reading-frame shifts and theconsequences which this entails regarding the formation of an intactprotein.

The mutagenic agents (mutagens) which can be used for generating randomor site-specific mutations, and the methods and techniques which can beapplied, are known to the skilled worker. Such methods and mutagens aredescribed for example in A. M. van Harten [(1998), “Mutation breeding:theory and practical applications”, Cambridge University Press,Cambridge, UK], E Friedberg, G Walker, W Siede [(1995), “DNA Repair andMutagenesis”, Blackwell Publishing], or K. Sankaranarayanan, J. M.Gentile, L. R. Ferguson [(2000) “Protocols in Mutagenesis”, ElsevierHealth Sciences].

Usual molecular-biological methods and processes, such as the in vitromutagenesis kit, LA PCR in vitro Mutagenesis Kit (Takara Shuzo, Kyoto),or PCR mutageneses using suitable primers may be employed forintroducing site-specific mutations.

As has already been mentioned above, a multiplicity of chemical,physical and biological mutagens exists.

Those mentioned hereinbelow are given by way of example, but not bylimitation.

Chemical mutagens can be distinguished by their mechanism of action.Thus, there are base analogs (for example 5-bromouracil, 2-aminopurine),mono- and bifunctional alkylating agents (for example monofunctionalagents such as ethylmethylsulfonate, dimethyl sulfate, or bifunctionalagents such as dichloroethyl sulfite, mitomycin,nitrosoguanidine-dialkylnitrosamine, N-nitrosoguanidine derivatives) orintercalating substances (for example acridine, ethidium bromide).

Physical mutagens are, for example, ionizing radiation. Ionizingradiation is electromagnetic waves or particle radiation capable ofionizing molecules, i.e. of removing electrons from the latter. Theremaining ions are highly reactive in most cases, so that, if they aregenerated in live tissue, are capable of causing great damage, forexample to the DNA, and (at low intensity) thereby inducing mutations.Ionizing radiation is, for example, gamma-radiation (photo energy ofapproximately one megaelectron volt MeV), X-rays (photo energy of aplurality of or many kiloelectron volts keV) or else ultraviolet light(UV light, photon energy of above 3.1 eV). UV light causes the formationof dimers between bases; with thymidine dimers, which give rise tomutations, being the most frequent here.

The traditional generation of mutants by treating the seeds withmutagenic agents such as, for example, ethylmethylsulfonate (EMS)(Birchler J A, Schwartz D. Biochem Genet. December; 17(11-12):1173-80;Hoffmann G R. Mutat Res. 1980 January; 75(1):63-129) or ionizingradiation has been joined by the use of biological mutagens, for exampletransposons (for example Tn5, Tn903, Tn916, Tn1000, Balcells et al.,1991, May B P et al. (2003) Proc Natl Acad Sci USA. September 30;100(20):11541-6.) or molecular-biological methods such as themutagenesis by means of T-DNA insertion (Feldman, K. A. Plant J.1:71-82.1991, Koncz et al. (1992) Plant Mol Biol 20(5):963-976).

The use of chemical or biological mutagens is preferred for thegeneration of mutated gene variants. In the case of chemical agents, thegeneration of mutants by use of EMS (ethylmethylsulfonate) mutagenesisis mentioned by particular preference. In the case of the generation ofmutants using biological mutagenesis, the T-DNA mutagenesis ortransposon mutagenesis may be mentioned by preference.

Thus, it is also possible to employ those polypeptides for the methodaccording to the invention which are obtained as the result of amutation of a polypeptide according to the invention, for example asshown in SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,30, 32, 34, 36, 38, 42, 44, 60, 61 or 62.

All substances and compounds which directly or indirectly bring about areduction in the polypeptide quantity, RNA quantity, gene activity orpolypeptide activity of an Armadillo repeat ARM1 protein will besummarized in this application under the term “anti-Armadillo repeatARM1 protein compounds”. The term “anti-Armadillo repeat ARM1 proteincompound” explicitly includes the nucleic acid sequences, peptides,proteins or other factors which are employed in the above-describedmethods.

In a further preferred embodiment of the present invention, an increasein the resistance to pathogens from the families Blumeriaceae,Pucciniaceae, Mycosphaerellaceae and Hypocreaceae in a monocotyledonousor dicotyledonous plant or an organ, tissue or a cell thereof isobtained by:

-   a) introduction, into a plant cell, of a recombinant expression    cassette comprising an “anti-Armadillo repeat ARM1 protein compound”    in operable linkage with a promoter which is active in plants;-   b) regeneration of the plant from the plant cell; and-   c) expression of said “anti-Armadillo repeat ARM1 protein compound”    in a sufficient quantity and over a sufficiently long period to    generate, or to increase, a pathogen resistance in said plant.

For example, regarding a nucleic acid sequence, an expression cassetteor a vector comprising said nucleic acid sequence or an organismtransformed with said nucleic acid sequence, expression cassette orvector, “transgenic” means all those constructs or organisms which arethe result of recombinant methods and in which either

-   a) the Armadillo repeat ARM1 protein nucleic acid sequence, or-   b) a genetic control sequence which is operably linked with the    Armadillo repeat ARM1 protein nucleic acid sequence, for example a    promoter, or-   c) (a) and (b)    are not in their natural genetic environment or have been modified    by recombinant methods, it being possible for the modification to be    for example a substitution, addition, deletion or insertion of one    or more nucleotide residue(s). Natural genetic environment means the    natural chromosomal locus in the original organism, or else the    presence in a genomic library. In the case of a genomic library, the    natural genetic environment of the nucleic acid sequence is    preferably retained, at least in part, the environment flanks the    nucleic acid sequence at least on one side and has a sequence length    of at least 50 bp, preferably at least 500 bp, especially preferably    at least 1000 bp, very especially preferably at least 5000 bp. A    naturally occurring expression cassette—for example the naturally    occurring combination of the Armadillo repeat ARM1 protein promoter    with the corresponding Armadillo repeat ARM1 protein gene—becomes a    transgenic expression cassette when the latter is modified by    non-natural, synthetic (“artificial”) methods, such as, for example,    treatment with a mutagen. Suitable methods are described (U.S. Pat.    No. 5,565,350; WO 00/15815).

For the purposes of the invention, “introduction” comprises all thosemethods which are suitable for introducing an “anti-Armadillo repeatARM1 protein compound” directly or indirectly into a plant or into acell, compartment, tissue, organ or seeds thereof, or for generatingsuch a compound therein. It comprises direct and indirect methods. Theintroduction can lead to a transient presence of one “anti-Armadillorepeat ARM1 protein compound” (for example of a dsRNA) or else to astable presence.

As the result of the differing nature of the above-described approaches,the “anti-Armadillo repeat ARM1 protein compound” can exert its functiondirectly (for example by insertion into an endogenous Armadillo repeatARM1 protein gene). However, the function can also be exerted indirectlyafter transcription into an RNA (for example in the case of antisenseapproaches) or after transcription and translation into a protein (forexample in the case of binding factors). Both direct and indirectlyacting “anti callose synthase compounds” are comprised in accordancewith the invention.

“Introduction” comprises, in the context of this description, in generalfor example methods such as transfection, transduction ortransformation.

Thus, “anti-Armadillo repeat ARM1 compound” also comprises for examplerecombinant expression constructs which bring about an expression (i.e.transcription and, if appropriate, translation) of, for example, anArmadillo repeat ARM1 protein dsRNA or an Armadillo repeat ARM1 protein“antisense” RNA, preferably in a plant or in a part, tissue, organ orseed thereof.

In said expression constructs/expression cassettes, a nucleic acidmolecule whose expression (transcription and, if appropriate,translation) generates an “anti-Armadillo repeat ARM1 protein compound”is preferably in operable linkage with at least one genetic controlelement (for example a promoter) which ensures an expression in plants.If the expression construct is to be introduced directly into the plantand the “anti-Armadillo repeat ARM1 protein compound” (for example theArmadillo repeat ARM1 protein dsRNA) is to be generated therein inp/ante, plant-specific genetic control elements (for example promoters)are preferred. However, the “anti-Armadillo repeat ARM1 proteincompound” can also be generated in other organisms or in vitro and thenbe introduced into the plant. Here, all prokaryotic or eukaryoticgenetic control elements (for example promoters) which permit theexpression in the respective plant which has been chosen for thegeneration are preferred.

An “operable” linkage is understood as meaning for example thesequential arrangement of a promoter with the nucleic acid sequence tobe expressed (for example an “anti-Armadillo repeat ARM1 proteincompound”) and, if appropriate, further regulatory elements such as, forexample, a terminator in such a way that each of the regulatory elementsis capable of fulfilling its function in the transgenic expression ofthe nucleic acid sequence, depending on the arrangement of the nucleicacid sequences to sense or antisense RNA. A direct linkage in thechemical sense is not necessarily required for this purpose. Geneticcontrol sequences such as, for example, enhancer sequences, can alsoexert their function on the target sequence from positions which arefurther removed or else from other DNA molecules. Preferred arrangementsare those in which the nucleic acid sequence to be expressedrecombinantly is positioned behind the sequence which acts as promoter,so that the two sequences are bonded covalently with one another. Inthis context, the distance between the promoter sequence and nucleicacid sequence to be expressed recombinantly is preferably less than 200base pairs, especially preferably less than 100 base pairs, veryespecially preferably less than 50 base pairs.

The preparation of a functional linkage and the preparation of anexpression cassette can be accomplished by means of customaryrecombination and cloning techniques as are described for example inManiatis. T, Fritsch E F and Sambrook J (1989) Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor(NY), in Silhavy T J, Berman M L and Enquist L W (1984) Experiments withGene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor (NY), inAusubel F M et al. (1987) Current Protocols in Molecular Biology, GreenePublishing Assoc. and Wiley Interscience and in Gelvin et al. (1990) in:Plant Molecular Biology Manual. However, it is also possible to positionfurther sequences which, for example, act as a linker with specificrestriction enzyme cleavage sites or as a signal peptide between the twosequences. Moreover, the insertion of sequences can lead to theexpression of fusion proteins. Preferably, the expression cassetteconsisting of a linkage of promoter and nucleic acid sequence to beexpressed can be present in vector-integrated form and can be insertedinto a plant genome by, for example, transformation.

However an expression cassette is also understood as meaning thoseconstructs in which a promoter is placed behind an element of choice,for example by a homologous recombination, for example an endogenousArmadillo repeat ARM1 protein gene, and, in said example, expression ofan antisense Armadillo repeat ARM1 protein RNA effects reductionaccording to the invention of an Armadillo repeat ARM1 protein.Similarly, it is also possible to place an element, for example an“anti-Armadillo repeat ARM1 protein compound” (for example a nucleicacid sequence coding for an Armadillo repeat ARM1 protein dsRNA or anArmadillo repeat ARM1 protein antisense RNA) behind an endogenouspromoter in such a way that the same effect occurs. Both approachesresult in expression cassettes for the purposes of the invention.

Plant-specific promoters means in principle any promoter which iscapable of controlling the expression of genes, in particular foreigngenes, in plants or plant parts, plant cells, plant tissues, plantcultures. Here, the expression can be for example constitutional,inducible or development-dependent.

The following promoters are preferred:

a) Constitutive Promoters

Preferred vectors are those which make possible a constitutiveexpression in plants (Benfey et al. (1989) EMBO J 8:2195-2202).“Constitutive” promoter means those promoters which ensure expression innumerous, preferably all, tissues over a relatively large period ofplant development, preferably at all times during plant development. Inparticular, a plant promoter or a promoter derived from a plant virus ispreferably used. The promoter of the 35S transcript of the CaMVcauliflower mosaic virus (Franck et al. (1980) Cell 21:285-294; Odell etal. (1985) Nature 313:810-812; Shewmaker et al. (1985) Virology140:281-288; Gardner et al. (1986) Plant Mol Biol 6:221-228) or the 19SCaMV Promoter (U.S. Pat. No. 5,352,605; WO 84/02913; Benfey et al.(1989) EMBO J 8:2195-2202) is particularly preferred. A further suitableconstitutive promoter is the rubisco small subunit (SSU) promoter (U.S.Pat. No. 4,962,028), the promoter of agrobacterium nopaline synthase,the TR double promoter, the agrobacterium OCS (octopine synthase)promoter, the ubiquitin promoter (Holtorf S et al. (1995) Plant Mol Biol29:637-649), the ubiquitin 1 promoter (Christensen et al. (1992) PlantMol Biol 18:675-689; Bruce et al. (1989) Proc Natl Acad Sci USA86:9692-9696), the Smas promoter, the cinnamyl-alcohol dehydrogenasepromoter (U.S. Pat. No. 5,683,439), the promoters of vacuolar ATPasesubunits or the promoter of a proline-rich protein from wheat (WO91/13991), and further promoters of genes whose constitutive expressionin plants is known to the skilled worker. Especially preferred asconstitutive promoter is the promoter of nitrilase-1 (nit1) gene from A.thaliana (GenBank Acc. No.: Y07648.2, Nukleotide 2456-4340, Hillebrandet al. (1996) Gene 170:197-200).

b) Tissue-Specific Promoters

Some embodiments employ promoters with specificities for the anthers,ovaries, flowers, leaves, stems, roots and seeds.

Seed-specific promoters such as, for example, the promoter of phaseolin(U.S. Pat. No. 5,504,200; Bustos M M et al. (1989) Plant Cell1(9):839-53), of the 2S albumin gene (Joseffson L G et al. (1987) J BiolChem 262:12196-12201), of legumin (Shirsat A et al. (1989) Mol Gen Genet215(2): 326-331), of the USP (unknown seed protein; Bäumlein H et al.(1991) Mol Gen Genet 225(3):459-67), of the napin gene (U.S. Pat. No.5,608,152; Stalberg K et al. (1996) L Planta 199:515-519), of sucrosebinding protein (WO 00/26388) or the legumin B4 promoter (LeB4; BäumleinH et al., (1991) Mol Gen Genet 225: 121-128; Baeumlein et al. (1992)Plant Journal 2(2):233-9; Fiedler U et al. (1995) Biotechnology (NY)13(10):1090f), the oleosin promoter from arabidopsis (WO 98/45461), theBce4 promoter from Brassica (WO 91/13980). Further suitableseed-specific promoters are those of the genes coding for the highmolecular weight glutenin (HMWG), gliadin, branching enzyme, ADP glucosepyrophosphatase (AGPase) or starch synthase. Further preferred promotersare those allowing seed-specific expression in monocotyledons such asmaize, barley, wheat, rye, rice etc. It is possible and advantageous toemploy the promoter of the Ipt2 or Ipt1 gene (WO 95/15389, WO 95/23230)or the promoters described in WO 99/16890 (promoters of the hordeingene, of the glutelin gene, of the oryzin gene, of the prolamin gene, ofthe gliadin gene, of the zein gene, of the kasirin gene or of thesecalin gene).

Tuber-, storage root- or root-specific promoters, for example thepatatin class I promoter (B33) or the promoter of the potato cathepsin Dinhibitor.

Leaf-specific promoters, for example for example the promoter of thecytosolic FBPase from potato (WO 97/05900), the SSU promoter (smallsubunit) of the rubisco (ribulose-1,5-bisphosphate carboxylase) or theST-LSI promoter from potato (Stockhaus et al. (1989) EMBO J8:2445-2451). Epidermis-specific promoters, for example the promoter ofthe OXLP gene (“oxalate oxidase like protein”; Wei et al. (1998) PlantMol. Biol. 36:101-112).

Examples of other tissue-specific promoters are:

Flower-Specific Promoters

for example the phytoen synthase promoter (WO 92/16635) or the promoterof the P-rr gene (WO 98/22593).

Anther-Specific Promoters

for example the 5126 promoter (U.S. Pat. No. 5,689,049, U.S. Pat. No.5,689,051), the glob-I promoter and the γ-zein promoter.

c) Chemically Inducible Promoters

The expression cassettes may also comprise a chemically induciblepromoter (review article: Gatz et al. (1997) Annu. Rev. Plant PhysiolPlant Mol Biol 48:89-108) through which expression of the exogenous genein the plant can be controlled at a particular point in time. Promotersof this type, such as, for example, the PRP1 promoter (Ward et al.(1993) Plant Mol Biol 22:361-366), a salicylic acid-inducible promoter(WO 95/19443), a benzenesulfonamide-inducible promoter (EP 0 388 186), atetracycline-inducible promoter (Gatz et al. (1992) Plant J 2:397-404),an abscisic acid-inducible promoter (EP 0 335 528) and an ethanol- orcyclohexanone-inducible promoter (WO 93/21334) can likewise be used.Thus, for example, the expression of a molecule which reduces orinhibits the Armadillo repeat ARM1 protein function, such as, forexample, the dsRNA, ribozymes, antisense nucleic acid molecules and thelike which have been listed above can be induced at suitable points intime.

d) Stress- or Pathogen-Inducible Promoters

Very especially advantageous is the use of inducible promoters forexpressing the RNAi constructs employed for reducing the callosesynthase polypeptide quantity, activity or function, which, for example,when pathogen-inducible promoters are used, makes possible an expressiononly when required, i.e. in the case of attack by pathogens).

In one embodiment, the method according to the invention therefore usespromoters which are active in plants which are pathogen-induciblepromoters.

Pathogen-inducible promoters comprise the promoters of genes which areinduced as a result of pathogen attack, such as, for example, genes ofPR proteins, SAR proteins, β-1,3-glucanase, chitinase, etc. (for exampleRedolfi et al. (1983) Neth J Plant Pathol 89:245-254; Uknes, et al.(1992) Plant Cell 4:645-656; Van Loon (1985) Plant Mol Viral 4:111-116;Marineau et al. (1987) Plant Mol Biol 9:335-342; Matton et al. (1987)Molecular Plant-Microbe Interactions 2:325-342; Somssich et al. (1986)Proc Natl Acad Sci USA 83:2427-2430; Somssich et al. (1988) Mol GenGenetics 2:93-98; Chen et al. (1996) Plant J 10:955-966; Zhang and Sing(1994) Proc Natl Acad Sci USA 91:2507-2511; Warner, et al. (1993) PlantJ 3:191-201; Siebertz et al. (1989) Plant Cell 1:961-968) (1989).

Also comprised are wound-inducible promoters such as that of the pinIIgene (Ryan (1990) Ann Rev Phytopath 28:425-449; Duan et al. (1996) NatBiotech 14:494-498), of the wun1 and wun2 gene (U.S. Pat. No.5,428,148), of the win1 and win2 gene (Stanford et al. (1989) Mol GenGenet 215:200-208), of the systemin gene (McGurl et al. (1992) Science225:1570-1573), of the WIP1 gene (Rohmeier et al. (1993) Plant Mol Biol22:783-792; Eckelkamp et al. (1993) FEBS Letters 323:73-76), of the MPIgene (Corderok et al. (1994) Plant J 6(2):141-150) and the like.

A source of further pathogen-inducible promoters is the PR gene family.A series of elements in these promoters have proved advantageous. Thus,the region −364 to −288 in the promoter of PR-2d mediates salicylatespecificity (Buchel et al. (1996) Plant Mol Biol 30, 493-504). Thesequence 5′-TCATCTTCTT-3′ occurs repeatedly in the promoter of thebarley β-1,3-glucanase and in more than 30 other stress-induced genes.In tobacco, this region binds a nuclear protein whose abundance isincreased by salicylate. The PR-1 promoters from tobacco and Arabidopsis(EP-A 0 332 104, WO 98/03536) are also suitable as pathogen-induciblepromoters Preferred, since particularly specifically induced bypathogens, are the “acidic PR-5”-(aPR5) promoters from barley (Schweizeret al. (1997) Plant Physiol 114:79-88) and wheat (Rebmann et al. (1991)Plant Mol Biol 16:329-331). aPR5 proteins accumulate withinapproximately 4 to 6 hours after attack by pathogens and only show verylittle background expression (WO 99/66057). One approach for obtainingan increased pathogen-induced specificity is the generation of syntheticpromoters from combinations of known pathogen-responsive elements(Rushton et al. (2002) Plant Cell 14, 749-762; WO 00/01830; WO99/66057). Other pathogen-inducible promoters from different species areknown to the skilled worker (EP-A 1 165 794; EP-A 1 062 356; EP-A 1 041148; EP-A 1 032 684).

Further pathogen-inducible promoters comprise the Flachs Fis1 promoter(WO 96/34949), the Vst1 promoter (Schubert et al. (1997) Plant Mol Biol34:417-426) and the tobacco EAS4 sesquiterpene cyclase promoter (U.S.Pat. No. 6,100,451).

Other preferred promoters are those which are induced by biotic orabiotic stress, such as, for example, the pathogen-inducible promoter ofthe PRP1 gene (or gst1 promoter), for example from potato (WO 96/28561;Ward et al. (1993) Plant Mol Biol 22:361-366), the heat-inducible hsp70or hsp80 promoter from tomato (U.S. Pat. No. 5,187,267), thechill-inducible alpha-amylase promoter from potato (WO 96/12814), thelight-inducible PPDK promoter or the wounding-inducible pinII promoter(EP-A 0 375 091).

e) Mesophyll-Tissue-Specific Promoters

In one embodiment, the method according to the invention employsmesophyll-tissue-specific promoters such as, for example, the promoterof the wheat germin 9f-3.8 gene (GenBank Acc.-No.: M63224) or the barleyGerA promoter (WO 02/057412). Said promoters are particularlyadvantageous since they are both mesophyll-tissue-specific andpathogen-inducible. Also suitable is the mesophyll-tissue-specificArabidopsis CAB-2 promoter (GenBank Acc. No.: X15222), and the Zea maysPPCZm1 promoter (GenBank Acc. No.: X63869) or homologs thereof.Mesophyll-tissue-specific means that the transcription of a gene islimited to as few as possible plant tissues which comprise the mesophylltissue as the result of the specific interaction of cis elements presentin the promoter sequence and transcription factors binding to theseelements; preferably, it means a transcription which is limited to themesophyll tissue.

As regards further promoters which are expressed essentially in themesophyll or in the epidermis, see the enumeration inserted furtherabove.

f) Development-Dependent Promoters

Examples of further suitable promoters are fruit ripening-specificpromoters such as, for example, the fruit ripening-specific promoterfrom tomato (WO 94/21794, EP 409 625). Development-dependent promotersinclude some of the tissue-specific promoters because the development ofindividual tissues naturally takes place in a development-dependentmanner.

Constitutive, and leaf- and/or stem-specific, pathogen-inducible,root-specific, mesophyll-tissue-specific promoters are particularlypreferred, with constitutive, pathogen-inducible,mesophyll-tissue-specific and root-specific promoters being mostpreferred.

A further possibility is for further promoters which make expressionpossible in further plant tissues or in other organisms such as, forexample, E. coli bacteria to be operably linked to the nucleic acidsequence to be expressed. All the promoters described above are inprinciple suitable as plant promoters.

Other promoters which are suitable for expression in plants aredescribed (Rogers et al. (1987) Meth in Enzymol 153:253-277; Schardl etal. (1987) Gene 61:1-11; Berger et al. (1989) Proc Natl Acad Sci USA86:8402-8406).

The nucleic acid sequences present in the expression cassettes orvectors of the invention may be operably linked to further geneticcontrol sequences besides a promoter. The term genetic control sequenceshas a wide meaning and means all sequences which have an influence onthe coming into existence or the function of the expression cassette ofthe invention. For example, genetic control sequences modifytranscription and translation in prokaryotic or eukaryotic organisms.The expression cassettes of the invention preferably comprise a promoterwith an abovementioned specificity 5-upstream from the particularnucleic acid sequence which is to be expressed transgenically, and aterminator sequence as additional genetic control sequence3′-downstream, and if appropriate further conventional regulatoryelements, in each case operably linked to the nucleic acid sequence tobe expressed transgenically.

Genetic control sequences also comprise further promoters, promoterelements or minimal promoters capable of modifying theexpression-controlling properties. It is thus possible for examplethrough genetic control sequences for tissue-specific expression to takeplace additionally dependent on particular stress factors. Correspondingelements are described for example for water stress, abscisic acid (LamE and Chua N H, J Biol Chem 1991; 266(26): 17131-17135) and heat stress(Schoffl F et al., Molecular & General Genetics 217(2-3):246-53, 1989).

It is possible in principle for all natural promoters with theirregulatory sequences like those mentioned above to be used for themethod of the invention. It is additionally possible also for syntheticpromoters to be used advantageously.

Genetic control sequences further comprise also the 5′-untranslatedregions, introns or noncoding 3′ region of genes such as, for example,the actin-1 intron, or the Adh1-S introns 1, 2 and 6 (generally: TheMaize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, NewYork (1994)). It has been shown that these may play a significantfunction in the regulation of gene expression. It has thus been shownthat 5′-untranslated sequences are capable of enhancing transientexpression of heterologous genes. An example of a translation enhancerwhich may be mentioned is the 5′ leader sequence from the tobacco mosaicvirus (Gailie et al. (1987) Nucl Acids Res 15:8693-8711) and the like.They may in addition promote tissue specificity (Rouster J et al. (1998)Plant J 15:435-440).

The expression cassette may advantageously comprise one or moreso-called enhancer sequences in operable linkage with the promoter,which make increased transgenic expression of the nucleic acid sequencepossible. Additional advantageous sequences such as further regulatoryelements or terminators can also be inserted at the 3′ end of thenucleic acid sequences to be expressed recombinantly. The nucleic acidsequences to be expressed recombinantly may be present in one or morecopies in the gene construct.

Polyadenylation signals suitable as control sequences are plantpolyadenylation signals, preferably those which correspond essentiallyto T-DNA polyadenylation signals from Agrobacterium tumefaciens, inparticular to gene 3 of the T-DNA (octopine synthase) of the Ti plasmidpTiACHS (Gielen et al. (1984) EMBO J 3:835 ff) or functional equivalentsthereof. Examples of particularly suitable terminator sequences are theOCS (octopine synthase) terminator and the NOS (nopaline synthase)terminator.

Control sequences additionally mean those which make homologousrecombination or insertion into the genome of a host organism possibleor allow deletion from the genome. In homologous recombination, forexample, the natural promoter of a particular gene can be specificallyreplaced by a promoter with specificity for the embryonal epidermisand/or the flower.

An expression cassette and/or the vectors derived from it may comprisefurther functional elements. The term functional element has a widemeaning and means all elements which have an influence on theproduction, replication or function of the expression cassettes, thevectors or the transgenic organisms of the invention. Non-restrictiveexamples which may be mentioned are:

-   a) Selection markers which confer a resistance to a metabolism    inhibitor such as 2 deoxyglucose 6-phosphate (WO 98/45456),    antibiotics or biocides, preferably herbicides, for example    kanamycin, G 418, bleomycin, hygromycin or phosphinotricin and the    like. Especially preferred selection markers are those which confer    a resistance to herbicides. DNA sequences which code for    phosphinothricin acetyltransferases (PAT), which inactivate    glutamine synthase inhibitors (bar and pat gene),    5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase genes)    which confer resistance to Glyphosat® (N-(phosphono-methyl)glycine),    the gox gene, which codes for the Glyphosat®-degrading enzyme    (glyphosate oxidoreductase), the deh gene (coding for a dehalogenase    which inactivates dalapon), sulfonylurea- and    imidazolinone-inactivating acetolactate synthases and bxn genes    which code for bromoxynil-degrading nitrilase enzymes, the aasa    gene, which confers a resistance to the antibiotic apectinomycin,    the streptomycin phosphotransferase (SPT) gene, which makes possible    a resistance to streptomycin, the neomycin phosphotransferase    (NPTII) gene, which confers a resistance to kanamycin or    geneticidin, the hygromycin phosphotransferase (HPT) gene, which    mediates a resistance to hygromycin, the acetolactate synthase gene    (ALS), which mediates a resistance to sulfonylurea herbicides (for    example mutated ALS variants with, for example, the S4 and/or Hra    mutation).-   b) Reporter genes which code for easily quantifiable proteins and    ensure via an intrinsic color or enzymic activity an assessment of    the transformation efficiency or of the location or timing of    expression. Very particular preference is given in this connection    to reporter proteins (Schenborn E, Groskreutz D. Mol Biotechnol.    1999; 13(1):29-44) such as the green fluorescence protein (GFP)    (Sheen et al. (1995) Plant Journal 8(5):777-784; Haselhoff et    al. (1997) Proc Natl Acad Sci. USA 94(6):2122-2127; Reichel et    al. (1996) Proc Natl Acad Sci USA 93(12):5888-5893; Tian et    al. (1997) Plant Cell Rep 16:267-271; WO 97/41228; Chui W L et    al. (1996) Curr Biol 6:325-330; Leffel S M et al. (1997)    Biotechniques. 23(5):912-8), the chloramphenicoltransferase, a    luciferase (Ow et al. (1986) Science 234:856-859; Millar et    al. (1992) Plant Mol Biol Rep 10:324-414), the aequorin gene    (Prasher et al. (1985) Biochem Biophys Res Commun 126(3):1259-1268),    the β-galactosidase, R-locus gene (code for a protein which    regulates the production of anthocyanin pigments (red coloration) in    plant tissue and thus makes possible the direct analysis of the    promoter activity without the addition of additional adjuvants or    chromogenic substrates; Dellaporta et al., In: Chromosome Structure    and Function: Impact of New Concepts, 18th Stadler Genetics    Symposium, 11:263-282, (1988), with β-glucuronidase being very    especially preferred (Jefferson et al., EMBO J. 1987, 6, 3901-3907).-   c) Origins of replication which ensure replication of the expression    cassettes or vectors of the invention in, for example, E. coli.    Examples which may be mentioned are OR1 (origin of DNA replication),    the pBR322 ori or the P15A ori (Sambrook et al.: Molecular Cloning.    A Laboratory Manual, 2^(nd) ed. Cold Spring Harbor Laboratory Press,    Cold Spring Harbor, N.Y., 1989).-   d) Elements which are necessary for agrobacterium-mediated plant    transformation such as, for example, the right or left border of the    T-DNA or the vir region.

To select successfully transformed cells, it is generally requiredadditionally to introduce a selectable marker which confers to thesuccessfully transformed cells a resistance to a biocide (for example aherbicide), a metabolism inhibitor such as 2 deoxyglucose 6-phosphate(WO 98/45456) or an antibiotic. The selection marker permits theselection of the transformed cells from untransformed cells (McCormicket al. (1986) Plant Cell Reports 5:81-84).

The introduction of an expression cassette according to the inventioninto an organism or into cells, tissues, organs, parts or seeds thereof(preferably into plants or plant cells, tissues, organs, parts or seeds)can advantageously be accomplished using vectors in which the expressioncassettes are present. The expression cassette can be introduced intothe vector (for example a plasmid) via a suitable restriction cleavagesite. The resulting plasmid is first introduced into E. coli. Correctlytransformed E. coli are selected, cultured, and the recombinant plasmidis obtained using methods known to the skilled worker. Restrictionanalysis and sequencing can be used for verifying the cloning step.

Examples of vectors can be plasmids, cosmids, phages, viruses or elseagrobacteria. In an advantageous embodiment, the introduction of theexpression cassette is accomplished by means of plasmid vectors.Preferred vectors are those which make possible a stable integration ofthe expression cassette into the host genome.

The generation of a transformed organism (or a transformed cell)requires the introduction of suitable DNA molecules, and thus of the RNAmolecules or proteins formed as the result of their gene expression,into the host cell in question.

A multiplicity of methods (Keown et al. (1990) Methods in Enzymology185:527-537) is available for this procedure, which is referred to astransformation (or transduction or transfection). Thus, DNA or RNA canbe introduced for example directly by means of microinjection or bybombardment with DNA-coated microparticles. Also, it is possible topermeabilize the cell chemically, for example with polyethylene glycol,so that the DNA can enter the cell by diffusion. Alternatively, the DNAcan be introduced by protoplast fusion with other DNA-comprising unitssuch as minicells, cells, lysosomes or liposomes. Another suitablemethod for introducing DNA is electroporation, where the cells arereversibly permeabilized by means of an electrical pulse. Suitablemethods are described (for example in Bilang et al. (1991) Gene100:247-250; Scheid et al. (1991) Mol Gen Genet 228:104-112; Guerche etal. (1987) Plant Science 52:111-116; Neuhause et al. (1987) Theor ApplGenet 75:30-36; Klein et al. (1987) Nature 327:70-73; Howell et al.(1980) Science 208:1265; Horsch et al. (1985) Science 227:1229-1231;DeBlock et al. (1989) Plant Physiology 91:694-701; Methods for PlantMolecular Biology (Weissbach and Weissbach, eds.) Academic Press Inc.(1988); and Methods in Plant Molecular Biology (Schuler and Zielinski,eds.) Academic Press Inc. (1989)).

In plants, the described methods for the transformation and regenerationof plants from plant tissues or plant cells for the transient or stabletransformation are used. Suitable methods are mainly the transformationof protoplasts by means of polyethylene-glycol-induced DNA uptake, thebiolistic method with the gene gun, i.e. the so-called particlebombardment method, electroporation, the incubation of dry embryos inDNA-comprising solution, and Microinjection.

In addition to these “direct” transformation techniques, atransformation can also be carried out by bacterial infection, forexample by means of Agrobacterium tumefaciens or Agrobacteriumrhizogenes. The methods are described for example in Horsch R B et al.(1985) Science 225: 1229f.

If agrobacteria are used, the expression cassette is to be integratedinto specific plasmids, either into a shuttle or intermediate vector orinto a binary vector. If a Ti or Ri plasmid is used for thetransformation, preferably at least the right border, but in most casespreferably the right and the left border, of the Ti or Ri plasmid T-DNAis linked as flanking region with the expression cassette to beintroduced.

It is preferred to use binary vectors. Binary vectors are capable ofreplicating both in E. coli and in Agrobacterium. As a rule, theycomprise a selection marker gene and a linker or polylinker flanked bythe right and left T-DNA border sequence. They can be transformeddirectly into Agrobacterium (Holsters et al. (1978) Mol Gen Genet163:181-187). The selection marker gene permits a selection oftransformed agrobacteria and is, for example, the nptII gene, whichconfers a resistance to kanamycin. The agrobacterium which acts as hostorganism in this case should already comprise a plasmid with the virregion. This is required for transferring the T-DNA to the plant cell.An agrobacterium thus transformed can be used for the transformation ofplant cells. The use of T-DNA for the transformation of plant cells hasbeen studied and described extensively (EP 120 516; Hoekema, in: TheBinary Plant Vector System, Offsetdrukkerij Kanters B.V., Alblasserdam,Chapter V, An et al. (1985) EMBO J 4:277-287). Various binary vectorsare known and in some cases commercially available, such as, forexample, pBI101.2 or pBIN19 (Clontech Laboratories, Inc. USA).

In the case of the injection or electroporation of DNA or RNA into plantcells, the plasmid used need not meet any particular requirements.Simple plasmids such as those from the pUC series can be used. If intactplants are to be regenerated from the transformed cells, it is necessaryfor an additional selectable marker gene to be located on the plasmid.

Stably transformed cells, i.e. those which comprise the introduced DNAintegrated into the DNA of the host cell, can be selected fromuntransformed cells when a selectable marker is a component of theintroduced DNA. For example, any gene which is capable of conferring aresistance to antibiotics or herbicides (such as kanamycin, G 418,bleomycin, hygromycin or phosphinothricin and the like) can act asmarker (see hereinabove). Transformed cells which express such a markergene are capable of surviving in the presence of concentrations of asuitable antibiotic or herbicide which kill an untransformed wild type.Examples are mentioned above and preferably comprise the bar gene, whichconfers resistance to the herbicide phosphinothricin (Rathore K S et al.(1993) Plant Mol Biol 21(5):871-884), the nptII gene, which confersresistance to kanamycin, the hpt gene, which confers resistance tohygromycin, or the EPSP gene, which confers resistance to the herbicideglyphosate. The selection marker permits the selection of transformedcells from untransformed cells (McCormick et al. (1986) Plant CellReports 5:81-84). The plants obtained can be bred and hybridized in thecustomary manner. Two or more generations should preferably be grown inorder to ensure that the genomic integration is stable and hereditary.

The abovementioned methods are described for example in Jenes B et al.(1993) Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1,Engineering and Utilization, edited by SD Kung and R Wu, Academic Press,p. 128-143 and in Potrykus (1991) Annu Rev Plant Physiol Plant MolecBiol 42:205-225). The construct to be expressed is preferably clonedinto a vector which is suitable for transforming Agrobacteriumtumefaciens, for example pBin19 (Bevan et al. (1984) Nucl Acids Res12:8711f).

As soon as a transformed plant cell has been generated, an intact plantcan be obtained using methods known to the skilled worker. Here, thestarting material is, for example, callus cultures. The development ofshoot and root can be induced in the known manner from these as yetundifferentiated cell lumps. The plantlets obtained can be potted on andbred.

The skilled worker is also familiar with methods of regenerating plantparts and intact plants from plant cells. For example, methods describedby Fennell et al. (1992) Plant Cell Rep. 11: 567-570: Stoeger et al(1995) Plant Cell Rep. 14:273-278; Jahne et al. (1994) Theor Appl Genet89:525-533 are used for this purpose.

The method according to the invention can advantageously be combinedwith other methods which bring about a pathogen resistance (for exampleto insects, fungi, bacteria, nematodes and the like), stress resistanceor another improvement of the plant's characteristics. Examples arementioned inter alia in Dunwell J M, Transgenic approaches to cropimprovement, J Exp Bot. 2000; 51 Spec No; pages 487-96.

In a preferred embodiment, the reduction of the function of an Armadillorepeat ARM1 protein in a plant is accomplished in combination with anincrease in the activity of a Bax inhibitor 1 protein. This can beeffected for example by expressing a nucleic acid sequence which codesfor a Bax inhibitor 1 protein, for example in the mesophyll tissueand/or root tissue.

In the method according to the invention, the Bax inhibitor 1 proteinsfrom Horoeum vulgare or Nicotiana tabacum are especially preferred.

Another subject matter of the invention relates to nucleic acidmolecules which comprise nucleic acid molecules coding for Armadillorepeat ARM1 proteins from barley as shown by the polynucleotides SEQ, IDNo: 1, and to the nucleic acid sequences which are complementarythereto, and to the sequences derived as the result of the degeneracy(degeneration) of the genetic code and to the nucleic acid moleculeswhich code for functional equivalents of the polypeptides as shown inSEQ. ID No. 1, the nucleic acid molecules not consisting of the SEQ IDNO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,39, 41 or 43.

Another subject matter of the invention relates to the Armadillo repeatARM1 protein from barley as shown in SEQ. ID No.: 2 or to one whichcomprises these sequences, and to functional equivalents thereof, whichdo not correspond to one of the SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 42 or 44.

Another subject matter of the invention relates to doublestranded RNAnucleic acid molecules (dsRNA molecule) which, when introduced into aplant (or into a cell, tissue, organ or seed thereof), bring about thereduction of an Armadillo repeat ARM1 protein, where the sense strand ofsaid dsRNA molecule has at least 30%, preferably at least 40%, 50%, 60%,70% or 80%, especially preferably at least 90%, very especiallypreferably 100%, homology with a nucleic acid molecule as shown in SEQID No: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33,35, 37, 39, 41 or 43, or to a fragment of at least 17 base pairs,preferably at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30base pairs, especially preferably at least 40, 50, 60, 70, 80 or 90 basepairs, very especially preferably at least 100, 200, 300 or 400 basepairs, most preferably at least 500, 600, 700, 800, 900, at least 1000,base pairs and which has at least 50%, 60%, 70% or 80%, especiallypreferably at least 90%, very especially preferably 100%, homology witha nucleic acid molecule as shown in SEQ ID No: 1, 3, 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 43 but do notcorrespond to SEQ ID NO: 3, 5, 7, 9, 11, 13, 15117, 19, 21, 23, 25, 27,29, 31, 33, 35, 37, 39, 41 or 43.

The double-stranded structure can be formed starting from a single,autocomplementary strand or starting from two complementary strands. Inan especially preferred embodiment, sense and antisense sequence arelinked by a linking sequence (linker) and can form for example a hairpinstructure. The linking sequence can very especially preferably be anintron, which is spliced out after the dsRNA has been synthesized.

The nucleic acid sequence coding for a dsRNA can comprise furtherelements, such as, for example, transcription termination signals orpolyadenylation signals.

A further subject matter of the invention relates to transgenicexpression cassettes which comprise one of the nucleic acid sequencesaccording to the invention. In the transgenic expression cassettesaccording to the invention, the nucleic acid sequence coding for theArmadillo repeat ARM1 proteins from barley, wheat and maize is linkedwith at least one genetic control element as defined above in such amanner that the expression (transcription and, if appropriate,translation) can be accomplished in a desired organism, preferablymonocotyledonous plants. Genetic control elements which are suitable forthis purpose are described above. The transgenic expression cassettescan also comprise further functional elements as defined above.

Such expression cassettes comprise for example a nucleic acid sequenceaccording to the invention, for example one which is essentiallyidentical to a nucleic acid molecule. SEQ ID No: 1, 3, 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 43, or afragment thereof according to the invention, where said nucleic acidsequence is preferably arranged in sense orientation or in antisenseorientation relative to a promoter and can therefore lead to theexpression of sense or antisense RNA, where said promoter is a promoterwhich is active in plants, preferably a promoter which is inducible bypathogen attack. Also comprised according to the invention aretransgenic vectors which comprise said transgenic expression cassettes.

Another subject matter of the invention relates to plants which, as theresult of natural processes or of artificial induction, comprise one ormore mutations in a nucleic acid molecule which comprises the nucleicacid sequence as shown in SEQ ID No: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 43, where said mutationbrings about a reduction in the activity, function or polypeptidequantity of a polypeptide encoded by the nucleic acid molecules as shownin SEQ ID No: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,33, 35, 37, 39, 41 or 43. For example a mutation prepared and identifiedby tilling.

Preferred in this context are plants which belong to the family Poaceae,especially preferred are plants selected among the plant genera Hordeum,Avena, Secale, Triticum, Sorghum, Zea, Saccharum and Oryza, veryespecially preferably plants selected from the species Hordeum vulgare(barley), Triticum aestivum (wheat), Triticum aestivum subsp. spelta(spelt), Triticale, Avena sativa (oats), Secale cereale (rye), Sorghumbicolor (sorghum), Zea mays (maize), Saccharum officinarum (sugar cane)and Oryza sativa (rice).

One embodiment of the invention therefore relates to a monocotyledonousorganism comprising a nucleic acid sequence according to the inventionwhich comprises a mutation which brings about, in the organisms or partsthereof, a reduction in the activity of one of the proteins encoded bythe nucleic acid molecules according to the invention. For example, themutation relates to one or more amino acid residues which are identifiedas being conserved or highly conserved in the consensus sequence shownin the figures.

Accordingly, another subject matter of the invention relates totransgenic plants, transformed with at least

-   a) one nucleic acid sequence, which comprises the nucleic acid    molecules as shown in SEQ ID No: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,    21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 43, the nucleic acid    sequences complementary thereto, and the nucleic acid molecules    which code for functional equivalents of the polypeptides as shown    in SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,    30, 32, 34, 36, 38, 40, 42, 44, 60, 61 or 62,-   b) one double-stranded RNA nucleic acid molecule (dsRNA molecule)    which brings about the reduction of an Armadillo repeat ARM1    protein, where the sense strand of said dsRNA molecule has at least    30%, preferably at least 40%, 50%, 60%, 70% or 80%, especially    preferably at least 90%, very especially preferably 100%, homology    with a nucleic acid molecule as shown in S SEQ ID No: 1, 3, 5, 7, 9,    11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or    43, or a fragment of at least 17 base pairs, preferably at least 18,    19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 base pairs,    especially preferably at least 40, 50, 60, 70, 80 or 90 base pairs,    very especially preferably at least 100, 200, 300 or 400 base pairs,    most preferably at least 500, 600, 700, 800, 900 or more base pairs,    which has at least 50%, 60%, 70% or 80%, especially preferably at    least 90%, very especially preferably 100%, homology with a nucleic    acid molecule as shown in SEQ ID No: 1, 3, 5, 7, 9, 11, 13, 15, 17,    19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 43,-   c) one transgenic expression cassette which comprises one of the    nucleic acid sequences according to the invention, or a vector    according to the invention, and cells, cell cultures, tissues,    parts—such as, for example in the case of plant organisms, leaves,    roots and the like—or propagation material derived from such    organisms,    where in one embodiment the nucleic acid molecules do not consist of    the nucleic acid molecules shown in SEQ ID No: 1, 3, 5, 7, 9, 11,    13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 43 and    in one embodiment do not consist of the polypeptide molecules shown    in SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,    30, 32, 34, 36, 38, 40, 42, 44, 60, 61 or 62 and

In one embodiment, the plant according to the invention or the plantused in accordance with the invention is not Arabidopsis thaliana.

Host or starting organisms which are preferred as “transgenic organisms”are mainly plants in accordance with the above definition. In oneembodiment, the transgenic organism is a mature plant, seed, shoot andseedling, and parts, propagation material and cultures derivedtherefrom, for example cell cultures. “Mature plants” means plants atany desired developmental stage beyond the seedling. “Seedling” means ayoung immature plant in an early developmental stage. Plants which areespecially preferred as host organisms are plants to which the methodaccording to the invention of obtaining a pathogen resistance inaccordance with abovementioned criteria can be applied. In oneembodiment, the plant is a monocotyledonous plant such as, for example,wheat, oats, sorghum and millet, barley, rye, maize, rice, buckwheat,sorghum, triticale, spelt or sugar cane, in particular selected from thespecies Hordeum vulgare (barley), Triticum aestivum (wheat), Triticumaestivum subsp. spelta (spelt), Triticale, Avena sativa (oats), Secalecereale (rye), Sorghum bicolor (sorghum), Zea mays (maize), Saccharumofficinarum (sugar cane) and Oryza sativa (rice).

The generation of the transgenic organisms can be accomplished with theabove-described methods for the transformation or transfection oforganisms.

Another subject matter of the invention relates to the transgenic plantsdescribed in accordance with the invention which additionally have anincreased Bax inhibitor 1 activity, with plants which have an increasedBax inhibitor 1 activity in mesophyll cells or root cells beingpreferred, with transgenic plants which belong to the family Poaceae andwhich have an increased Bax inhibitor 1 activity in mesophyll cells orroot cells being especially preferred, with transgenic plants selectedamong the plant genera Hordeum, Avena, Secale, Triticum, Sorghum, Zea,Saccharum and Oryza being even more preferred, and with the plantspecies Hordeum vulgare (barley), Triticum aestivum (wheat), Triticumaestivum subsp. spelta (spelt), Triticale, Avena sativa (oats), Secalecereale (rye), Sorghum bicolor (sorghum), Zea mays (maize), Saccharumofficinarum (sugar cane) and Oryza sativa (rice) being preferred most ofall.

Another subject matter of the invention relates to the use of thetransgenic organisms according to the invention and of the cells, cellcultures, parts—such as, for example in the case of transgenic plantorganisms, roots, leaves and the like—and transgenic propagationmaterial such as seeds or fruits derived therefrom for the preparationof foodstuffs or feedstuffs, pharmaceuticals or fine chemicals.

In one embodiment, the invention furthermore relates to a method for therecombinant production of pharmaceuticals or fine chemicals in hostorganisms, where a host organism or a part thereof is transformed withone of the above-described nucleic acid molecule expression cassettesand this expression cassette comprises one or more structural geneswhich code for the desired fine chemical or catalyze the biosynthesis ofthe desired fine chemical, where the transformed host organism is grownand where the desired fine chemical is isolated from the growth medium.This method can be applied widely to fine chemicals such as enzymes,vitamins, amino acids, sugars, fatty acids, natural and syntheticflavorings, aroma substances and colorants. Especially preferred is theproduction of tocopherols and tocotrienols and carotenoids. The growingof the transformed host organisms and the isolation from the hostorganisms or the growth medium are accomplished by methods known to theskilled worker. The production of pharmaceuticals such as, for example,antibodies or vaccines, is described in Hood E E, Jilka J M (1999). CurrOpin Biotechnol. 10(4):382-6; Ma J K, Vine N D (1999). Curr TopMicrobiol Immunol. 236:275-92.

In accordance with the invention, the expression of a structural genecan, of course, also take place, or be influenced, independently ofcarrying out the method according to the invention or using the subjectmatters according to the invention.

Sequences

-   1. SEQ ID NO: 1 and 2: HvArm-   2. SEQ ID NO: 3 and 4: OS_(—)1_XM_(—)479734.1-   3. SEQ ID NO: 5 and 6 Os2_XM_(—)463544-   4. SEQ ID NO: 7 and 8 Os_(—)3_AP003561-   5. SEQ ID NO: 9 and 10 Os_(—)4_XM_(—)506432-   6. SEQ ID NO: 11 and 12 NT_(—)1_AY219234-   7. SEQ ID NO: 13 and 14 At_(—)1_NM_(—)127878-   8. SEQ ID NO: 15 and 16 At_(—)2_AC004401-   9. SEQ ID NO: 17 and 18 At_(—)3_BT020206-   10. SEQ ID NO: 19 and 20 At_(—)4_AB007645-   11. SEQ ID NO: 21 and 22 At_(—)5_NM135336 (At3g54790)-   12. SEQ ID NO: 23 and 24 At_(—)6_AK118613-   13. SEQ ID NO: 25 and 26 At_(—)7_AL138650-   14. SEQ ID NO: 27 and 28 At_(—)8_AL133314-   15. SEQ ID NO: 29 and 30 At_(—)9_AC010870-   16. SEQ ID NO: 31 and 32 At_(—)10_AY125543 (At3g01400)-   17. SEQ ID NO:33 and 34 At_(—)11_AY087360-   18. SEQ ID NO: 35 and 36 At_(—)12_AB1016888-   19. SEQ ID NO: 37 and 38 At_(—)13_AK175585-   20. SEQ ID NO: 39 and 40 At_(—)14_AL049655-   21. SEQ ID NO:41 and 42 At_(—)15_AY096530 (At3g54850)-   22. SEQ ID NO: 43 and 44 At_(—)16_AK118730 (At4g16490)-   23. SEQ ID NO: 45 to 59: primers-   24. SEQ ID NO: 60, 61, 63: consensus sequences of polynucleotide SEQ    ID NO. from 1. to 22.

In the figures:

FIG. 1 (12 pages): depicts ARM1 nucleic acid sequences from barley,rice, and Arabidopsis thaliana.

FIG. 2 (6 pages): depicts ARM1 polypeptide sequences from barley, rice,and Arabidopsis thaliana.

FIG. 3 (20 pages): depicts a sequence alignment of ARM1 proteinsequences polypeptides from barley, rice, and Arabidopsis thaliana.

FIG. 4 (1 page): depicts increase in mildew resistance of barley due toRNAi of ARM repeat proteins

FIG. 5 (2 pages): depicts consensus sequences of the sequence alignmentof ARM1 protein sequences polypeptides from barley, rice, andArabidopsis thaliana.

EXAMPLES General Methods

The chemical synthesis of oligonucleotides can take place for example ina known manner by the phosphoamidite method (Voet, Voet, 2nd edition,Wiley Press New York, page 896-897). The cloning steps carried out forthe purposes of the present invention, such as, for example, restrictioncleavages, agarose gel electrophoresis, purification of DNA fragments,transfer of nucleic acids to nitrocellulose and nylon membranes, linkageof DNA fragments, transformation of E. coli cells, culturing ofbacteria, replication of phages and sequence analysis of recombinant DNAare carried out as described in Sambrook et al. (1989) Cold SpringHarbor Laboratory Press; ISBN 0-87969-309-6. The sequencing ofrecombinant DNA molecules takes place using a laser fluorescence DNAsequencer from the company MWG-Licor by the method of Sanger (Sanger etal. (1977) Proc Natl Acad Sci USA 74:5463-5467).

Example 1 Plants, Pathogens and Inoculation

The barley variety Golden Promise is from Patrick Schweizer, Institutfür Pflanzengenetik und Kulturpflanzenforschung Gatersleben. The varietyPallas and the backcrossed line BCIngrid-mlo5 was provided by Lisa Munk,Department of Plant Pathology, Royal Veterinary and AgriculturalUniversity, Copenhagen, Denmark. Its preparation is described (Kølster Pet al. (1986) Crop Sci 26: 903-907).

Unless otherwise described, the seed which has been pregerminated for 12to 36 hours in the dark on moist filter paper is placed in batches of 5grains along the edge of a square pot (8×8 cm) in Fruhstorfer soil typeP, covered with soil and watered regularly with tap water. All plantsare grown in controlled-environment cabinets or chambers at from 16 to18° C. for 5 to 8 days, at a relative atmospheric humidity of from 50 to60% and in a 16/8-hour photo period with 3000 and 5000 lux, respectively(50 and 60 μmols-¹m-² photon flux density, respectively) and employed inthe experiments in the seedling stage. In the case of experiments whereprimary leaves are treated, the latter are fully developed.

Before the plants are subjected to the transient transfectionexperiments, they are grown in controlled-environment cabinets orchambers at a daytime temperature of 24° C., night-time temperature of20° C., relative atmospheric humidity of 50 to 60% and a 16/8-hour photoperiod with 30 000 lux.

Powdery mildew of barley Blumeria graminis (DC) Speer fsp. hordei Em.Marchal der Rasse A6 (Wiberg A (1974) Hereditas 77: 89-148) (BghA6) isused to inoculate barley plants. The mildew was provided by the Institutfür Biometrie, JLU Gieβen. The inoculum is maintained incontrolled-environment cabinets under conditions which are identical tothose which have been described above for the plants by transferring theconidia from infected plant material to 7-day old barley plants cv.Golden Promise which have been raised at regular intervals, at a densityof 100 conidia/mm².

The inoculation with BghA6 is carried out using 7-day-old seedlings byshaking the conidia of infected plants in an inoculation tower at adensity of approximately 100 conidia/mm² (unless otherwise stated).

Example 2 RNA Extraction

Total RNA is extracted from 8 to 10 primary leaf segments (5 cm inlength) by means of “RNA extraction buffer” (AGS, Heidelberg, Germany).

To this end, central primary leaf segments 5 cm in length are harvestedand homogenized in liquid nitrogen using a pestle and mortar. Thehomogenate is stored at −70° C. until the RNA is extracted.

Total RNA is extracted from the frozen leaf material with the aid of anRNA extraction kit (AGS, Heidelberg). To this end, 200 mg of the frozenleaf material is covered with 1.7 ml of RNA extraction buffer (AGS) in amicrocentrifuge tube (2 ml) and immediately subjected to thoroughmixing. After the addition of 200 μl of chloroform, the mixture is againmixed thoroughly and shaken for 45 minutes at room temperature on anorbital shaker at 200 rpm. Thereafter, the mixture is centrifuged for 15minutes at 20 000 g and 4° C. in order to separate the phases, theaqueous top phase is transferred into a fresh microcentrifuge tube, andthe bottom phase is discarded. The aqueous phase is again purified with900 μl of chloroform by homogenizing 3 times for 10 seconds andrecentrifuging (see above) and removing the top phase. To precipitatethe RNA, 850 μl of 2-propanol are then added, the mixture is homogenizedand placed on ice for 30 to 60 minutes. Thereafter, the mixture iscentrifruged for 20 minutes (see above), the supernatant is carefullydecanted off, 2 ml of 70% strength ethanol (−20° C.) are added, using apipette, and the batch is mixed and again centrifuged for 10 minutes.The supernatant is then again decanted off and the pellet is carefullyfreed from residual fluid, using a pipette, and then dried in a streamof pure air on a sterile workbench. Thereafter, the RNA is dissolved in50 μl of DEPC water on ice, and the batch is mixed and centrifuged for 5minutes (see above) 40 μl of the supernatant are transferred into afresh microcentrifuge tube as RNA solution and stored at −70° C.

The RNA concentration is determined photometrically. To this end, theRNA solution is diluted 1:99 (v/v) with distilled water and theabsorbance (Photometer DU 7400, Beckman) is measured at 260 nm(E_(260 nm)=1 at 40 μg RNA/ml). In accordance with the calculated RNAcontents, the concentrations of the RNA solutions are subsequentlystandardized with DEPC water to 1 μg/μl and verified in an agarose gel.

To verify the RNA concentrations in a horizontal agarose gel (1% agarosein 1×MOPS buffer with 0.2 μg/ml ethidium bromide), 1 μl of RNA solutionis treated with 1 μl of 10×MO PS, 1 μl of color marker and 7 μl of DEPCwater, separated according to size at a voltage of 120 V in the gel in1×MOPS running buffer in the course of 1.5 hours and photographed underUV light. Any differences in concentration of the RNA extracts arestandardized with DEPC water, and the standardization is again verifiedin the gel.

Example 3 Cloning the Barley HvARM cDNA Sequence

The cDNA fragments required for isolating, cloning and sequencingarmadillo cDNA were obtained by means of RT PCR using the GeneRacer kit(Invitrogen Life Technologies). For this purpose, total RNA from barleyepidermis was used as template. RNA was isolated from epidermal cells ofIngrid+Bgt barley 12 h and 24 h after infection.

The HvArm cDNA sequence was extended by means of the RACE technologyusing the GeneRacer kit (INVITROGEN Life Technologies). For thispurpose, 4000 ng of total mRNA, 1 μl of 10×CIP buffer, 10 units of RNAseinhibitor, 10 units of CIP (calf intestinal phosphatase) andDEPC-treated water to a total volume of 10 μl were treated at 50° C. for1 h. The RNA was precipitated by adding a further 90 μl of DEPC waterand 100 μl of phenol:chloroform and mixing thoroughly for approx. 30sec. After centrifugation at 20 000 g for 5 min, the upper phase wasadmixed with 2 μl of 10 mg/ml mussel glycogen, 10 μl of 3 M sodiumacetate (pH 5.2) in a new micro-reaction vessel. The mixture was treatedwith 220 μl of 95% ethanol and incubated on ice. RNA was subsequentlyprecipitated by centrifugation at 20 000 g and 4° C. for 20 min. Thesupernatant was discarded, 500 μl of 75% ethanol were added, the mixturewas briefly vortexed and again centrifuged for 2 min (20 000 g). Thesupernatant was again discarded, the precipitate was dried in air atroom temperature for 2 min and subsequently suspended in 6 μl of DEPCwater. mRNA CAP structures were removed by adding 1 μl of 10×TAP buffer,10 units of RNAsin and 1 unit of TAP (tobacco acid pyrophosphatase). Themixture was incubated at 37° C. for 1 h and then cooled on ice. The RNAwas again precipitated, as described above, and transferred to areaction vessel containing 0.25 μg of GeneRacer oligonucleotide primer.The oligonucleotide primer was resuspended in the RNA solution, themixture was incubated at 70° C. for 5 min and then cooled on ice. Tothis 1 μl of 10× ligase buffer, 10 mM ATP, 1 unit of RNAsin and 5 unitsof T4 RNA ligase were added and the reaction mixture was incubated at37° C. for 1 h. The RNA was again precipitated, as described above, andresuspended in 7 μl of DEPC water. The RNA was admixed with 10 pmolGeneRacer Oligo-dT primer and 2 μl of each dNTP solution (25 mM), themixture was heated to 70° C. for 10 min and then again cooled on ice.This was followed by adding a mix of 2 μl of 10×RT buffer, 4 μl of 25 mMMgCl₂, 2 μl of 0.1M DTT, 5 U (1 μl) of SuperscriptIII transcriptase (200U/μl) and 1 μl RNAse Out (40 U/μl), incubating the reaction solution at50° C. for 50 min and then inactivating it at 85° C. for 5 min. Afterincubating with 1 μl RNAse H (2 U/μl) at 37° C. for 20 min, the firststrand cDNA prepared in this way was stored at −20° C.

The following primers were used for the RT PCR:

GeneRacer Oligo-dT Primer (Invitrogen Life Technologies):

(Seq ID No.: 45) GCTGTCAACGATACGCTACGTAACGGCATGACAGTG (T)18

For each reaction (total volume: 20 μL) 4000 ng of total RNA, 10 mMdNTPs, 50 μM GeneRacer Oligo-dT primer (Invitrogen Life Technologies), 1μl of RNase inhibitor and 1 μl of enzyme mix in 1×RT buffer (GeneRacerKit Invitrogen) were used.

The reaction was incubated at 50° C. for 50 minutes.

The subsequent primers were used for amplifying the 5, cDNA ends:

MWG 1: 5′GCAGACATGACCCAATCTTGGCAGG 3′ (Seq ID No.: 46) GR 5′ primer(Invitrogen): 5′cgactggagcacgaggacactga 3′ (Seq ID No.: 47) MWG 2:5′CCACGGTCAGCAACCTCTCCAGACG 3′ (Seq ID No.: 48) GeneRacer 5′nestedprimer (Invitrogen): 5′ggacactgacatggactgaaggagta 3′ (Seq ID No.: 49)MWG 3: 5′ cagatgatagttattgttgttgactgg 3′ (Seq ID No.: 50) GR 3′ primer(Invitrogen): 5′ GCTGTCAACGATACGCTACGTAACG 3′ (Seq ID No.: 51) MWG 4:5′ctcatcttctcaagctactggtgg 3′ (Seq ID No.: 52) GeneRacer 3′nested primer(Invitrogen): 5′CGCTACGTAACGGCATGACAGTG 3′ (Seq ID No.: 53)

The mixture (total volume: 50 μL) was composed as follows:

4 μl of MWG1 (10 pmol/μl)4.5 μl of 5′Gene Racer (10 pmol/μl)5 μl of 10× buffer Roche1.5 μl of 10 mM dNTPs1 μl of cDNA

1 μl of Taq (Roche) 33 μl of H2O

The following temperature program was used (GeneAmp PCR System 9700;Applied Biosystems):

94° C., 2 min denaturation  5 cycles of 94° C., 30 sec (denaturation)72° C., 2 min (extension)  5 cycles of 94° C., 30 sec (denaturation) 70°C., 2 min (extension) 30 cycles of 94° C., 30 sec (denaturation) 65° C.,30 sec (annealing) 68° C., 2 min (extension) 68° C., 7 min finalextension  4° C., cooling until further processing

The PCR did not produce any product. Starting from this, a nested RACEwith MWG2, the armadillo-specific oligonucleotide primer and theGeneRacer Nested 5′ primer was carried out:

94° C., 2 min denaturation 30 cycles of 94° C., 30 sec (denaturation)65° C., 30 sec (annealing) 68° C., 2 min (extension) 68° C., 7 min finalextension  4° C., cooling until further processing

The PCR resulted in a product of approx. 850 bp. The PCR productobtained was isolated via a 1% agarose gel, extracted from the gel andcloned into pCR4-Topo (Invitrogen Life Technologies) by means ofT-overhang ligation and sequenced. The sequence depicted under SEQ IDNO: is also identical to the barley armadillo sequence.

The full length HvArm sequence was amplified using the followingprimers:

MWG 29: 5′atatgcaaatggctctgctag 3′ (Seq ID No.: 54) MWG 30:5′TATCATCTCCTTCCCGAGTTC 3′ (Seq ID No.: 55)

The mixture (total volume: 50 μL) was composed as follows:

4 μl of MWG29 (10 pmol/μl)4 μl of MWG30 (10 pmol/μl)5 μl of 10×Pfu Ultra buffer (Stratagene)1.5 μl of 10 μM dNTPs1 μl of cDNA

1 μl of Pfu Ultra (Stratagene) 33 μl of H2O

The following temperature program was used (GeneAmp PCR System 9700;Applied Biosystems):

94° C., 2 min denaturation 30 cycles of 94° C., 30 sec (denaturation)55° C., 30 sec (annealing) 72° C., 1.5 min (extension) 72° C., 7 min,final extension  4° C., cooling until further processing

The PCR resulted in a product of 1326 bp. The PCR product obtained wasisolated via a 1% agarose gel, extracted from the gel and cloned intopCR4-Topo (Invitrogen Life Technologies) by means of T-overhang ligationand sequenced. The sequence depicted under SEQ ID NO: is also identicalto the barley armadillo sequence.

Example 4 Cloning of the Full Length cDNA Sequence of Arabidopsisthaliana AtARM (At2g23140)

The full length AtArm sequence was amplified using the followingprimers:

MWG 31: (Seq ID No.: 56) 5′ cccgggatgattttgcggttttggcgg 3′ MWG 32: (SeqID No.: 57) 5′ CCCGGGTCACAAGACAAAACATAAAAATAGG 3′ MWG 32b: (Seq ID No.:58) 5′gactcacactactctaatacc 3′ MWG 33: (Seq ID No.: 59)5′GACATCGTTTGTCTCACACC 3′

The mixture (total volume: 50 μL) was composed as follows (due to itssize of 2775 bp, the gene was divided into two parts for the PCR):

4 μl of MWG31 (10 pmol/μl)4 μl of MWG34 (10 pmol/μl)5 μl of 10×Pfu Ultra buffer (Stratagene)1.5 μl of 10 mM dNTPs1 μl of cDNA

1 μl of Pfu Ultra (Stratagene) 33 μl of H2O

and4 μl of MWG32 (10 pmol/μl)4 μl of MWC33 (10 pmol/μl)5 μl of 10×Pfu Ultra buffer (Stratagene)1.5 μl of 10 mM dNTPs1 μl of cDNA

1 μl of Pfu Ultra (Stratagene) 33 μl of H2O

The following temperature program was used (GeneAmp PCR System 9700;Applied Biosystems):

94° C., 2 min denaturation 30 cycles of 94° C., 30 sec (denaturation)59° C., 30 sec (annealing) 72° C., 1.5 min (extension) 72° C., 7 minfinal extension  4° C., cooling until further processing

The PCR results in a product of 1143 bp and 1705 bp, respectively. ThePCR product obtained is isolated via a 1% agarose gel, extracted fromthe gel and cloned into pCR4-Topo (Invitrogen Life Technologies) bymeans of T-overhang ligation and sequenced. The sequence depicted underSEQ ID NO: is also identical to the Arabidopsis thaliana armadillosequence.

In order to assemble the gene, the 1705 bp PCR product is cloned intopUC18. This is followed by cloning AtArm (1143 bp) into pUC18-AtArm(1705 bp).

An antisense construct is generated for constitutive expression. To thisend, HvArm antisense is cloned into the binary vector 1bxSuperGus byexcising HvArm via SmaI from pUC18 and cloning it via said cleavagesites into the 5′-terminally blunted 1 bxSuperGus (SacI/SmaI). Theorientation is verified by means of a test digest.

Example 5 Carrying Out the Transient Single-Cell RNAi AnalysisBiological Material

Barley near-isogenic lines (NiLs) of the cultivars cv Ingrid (Mlo) andIngrid BC₇ mlo5 or barley cv Golden Promise were grown incontrolled-environment chambers in pots filled with potting compost(provenance: IPK Gatersleben) (16 hours light from metal halogen lamps;8 hours darkness, relative atmospheric humidity of 70%, constanttemperature of 18° C.). Blumeria graminis DC Speer f.sp. hordei (Bgh)(isolate 4.8 comprising AvrMla9) was grown at 22° C. and 16 hours lightby weekly transfer to fresh barley leaves of the cultivar cv. GoldenPromise. Blumeria graminis DC Speer f.sp. tritici Em Marchal (Bgt) ofthe Swiss isolate FAL (Reckenholz) was propagated at 22° C. and 16 hourslight by weekly transfer to fresh leaves of wheat of the cultivar cv.Kanzler.

Plasmid Vectors

The vector pIPKTA38 was used as entry vector for the Gateway™ cloningsystem (Invitrogen). The vector is a pENTR1a derivative where the ccdBgene had been removed and a novel multiple cloning site had beeninserted. The destination vector used was pIPKTA30N, which is based on apUC18 background and which comprises a constitutive promoter, terminatorand two Gateway cassettes comprising attr sites, ccdB gene and achloramphenicol resistance gene. The two cassettes are arranged inopposite directions and separated from one another by a spacer from thewheat RGA2 gene (accession number AF326781). This vector system permitsa one-step transfer of two copies of a PCR fragment via entry vectorinto the dsRNAi vector by means of Gateway LR clonase reaction(Invitrogen).

PCR and Primer Design

EST sequences of the target gene were amplified via PCR. Purified DNAfrom the selected cDNA clones was used as template for the PCR reaction.The primers were derived with the aid of the software package “Primer3”in the batch-file mode using the 5′-EST sequences. The EST sequenceswere typically amplified with a universal forward primer and a reverseEST-specific primer. The amplificates were in the range of from 400-700bp. The primers were 20-22 bp in length and had a T_(m) of approx. 65°C. The PCR reactions were carried out in 96-well microtiter plates usinga DNA polymerase which produces blunt ends (ThermalAce; Invitrogen). ThePCR products were purified with the aid of the MinElute UF Kit (Qiagen,Hilden, Germany) and eluted with 25 μl of water.

Ligation into the Entry Vector

The PCR fragments were cloned into the Swa I cleavage site of thisvector pIPKTA38. The ligation was carried out at 25° C. in the presenceof the N U T4 DNA ligase (MBI Fermentas) and 5 U of Swa I per reaction.To optimize the reaction conditions for Swa I, the buffer wassupplemented with NaCl to a final concentration of 0.05 M. After 1 h,the reaction was terminated by heating for 15 minutes at 65° C.Thereafter, an additional 5 U of Swa I were added in order to suppress areligation of the plasmid. The Swa I buffer was supplemented withadditional NaCl to a final concentration of 0.1 M. The reaction mixtureswere incubated for a further hour at 25° C.

The resulting recombinant pIPKTA38-EST clones were employed for thetransformation of chemically competent E. coli DH10B cells in 96-wellPCR microtiter plates (5 μl of ligation mixture per 20 μl of competentcells) and plated onto LB agar plates with kanamycin. One colony of eachcloning reaction was picked and taken up in 1.2 ml of LB+kanamycinliquid culture and distributed in 96-deep-well plates. The plates werecovered with an air-permeable film and incubated for 18 hours at 37° C.on a shaker. Thereupon, the deep-well plates were centrifuged for 10minutes at 750 g, and the pellets were used for isolating the plasmid bymeans of the NucleoSpin Robot-96 plasmid kit (Macherey-Nagel). Thepresence of the pIPKTA38 plasmid was verified via restriction digestwith EcoRI. The positive pIPKTA38 clones were employed as donor vectorin the LR reaction.

LR Reaction and Preparation of RNAi Constructs

EST fragments in pIPKTA38 were cloned as inverted repeats into the RNAidestination vector pIPKTA30N via a single LR recombination reaction. Thereaction volume was reduced to 6 μl and comprised 1 μl of the pIPKTA38donor clone, 1 μl pIPKTA30N destination vector (150 ng/μl), 0.8 μL LRclonase enzyme mix and 3.2 μl of H₂O. The reaction was incubatedovernight at room temperature, and 5 μl of it were transformed into 20μl of chemically competent E. coli cells in 96-well PCR plates. Two96-deep-well plates with LB+ampicillin were half-filled with half thevolume of the transformation mix, sealed with an air-permeable film andincubated for 24 hours at 37° C. on a plate shaker. Thereafter, thedeep-well plates were centrifuged for 10 minutes at 750 g, and thepellets of two duplicates of each clone were combined and subjected tothe plasmid preparation. The NucleoSpin Robot-96 plasmid kit(Macherey-Nagel) was used for this purpose. The DNA quantity was onaverage 20-30 μg of DNA per clone.

Particle Bombardment and Inoculation with Fungal Spores

Segments of primary leaves of 7-day-old barley seedlings were placed on0.5% w/v Phytoagar (Ducheva) in water comprising 20 ppm of benzimidazoleand bombarded with gold particles (diameter 1 μm) in a PDS-1000/Hesystem (Bio-Rad, Munich, Germany) using the Hepta adaptor with a heliumpressure of 900 psi. Seven leaf segments were employed per bombardment.The particle coating with 0.5 M Ca(NO₃)₂ was carried out as described bySchweizer et al., 1999, except that the stock solution comprised 25 mgml⁻¹ gold. After the coating, all of the supernatant was removed, andthe particles were resuspended in 30 μl of pure ethanol. 2.18 mg of goldmicrocarrier were employed per bombardment. Four hours after thebombardment, the leaf segments were placed on 1% w/v Phytoagar (Ducheva)in water comprising 20 ppm of benzimidazole in 20×20 cm plates andweighted down at both ends.

The leaf segments were inoculated with spores of Bgt and Bgh 48 hours or96 hours after the particle bombardment. The plasmid pUbiGUS, whichcomprises the β-glucuronicse (GUS) gene under the control of the maizeubiquitin promoter, was employed as reporter construct for transformedepidermal cells. 40 hours after the inoculation, the leaf segments werestained on GUS activity and destained for 5 minutes with 7.5% w/vtrichloroacetic acid and 50% methanol. The GUS staining solution hasbeen described in Schweizer et al. 1999.

To evaluate the interaction of phenotypes, GUS-stained cells werecounted under an optical microscope, and the number of haustoria inthese transformed cells was determined, whereby the haustorial index isderived. As an alternative, the number of GUS-stained cells whichcomprised at least one haustorium was determined and the susceptibilityindex was calculated thereby.

Results: Increase in mildew resistance of barley due to RNAi of ARMrepeat proteins

Susceptibility Spores/mm² 0.08 200 0.07 0   0.04 0.01 0.06 0.08 0.07 2000.13 0.01 0.02 0.01 0.07 0.07 0.1  200 0.06 0.01 0.05 0.06 0.08 Rel HAUIndex (%) GUS Construct Mean SDM Cells TA30 (control) 100 0 5499 HO14H1848.57 10.03 1572 TA36 (Mlo RNAi) 8.88888889 0.88377212 2024

See also FIG. 4.

1. A method of increasing the resistance to pathogens in a plant or in apart of a plant, wherein comprising reducing the activity of anArmadillo repeat ARM1 protein in a plant or in a part of the plant. 2.The method according to claim 1, wherein the activity in the mesophyllcells and/or epidermal cells is reduced.
 3. The method according toclaim 1, wherein the polypeptide is encoded by a polynucleotidecomprising at least one nucleic acid molecule selected from the groupconsisting of: a) a nucleic acid molecule which codes for at least onepolypeptide comprising the sequence as shown in SEQ ID NO: 2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,60, 61 or 62; b) a nucleic acid molecule which comprises at least onepolynucleotide of the sequence as shown in SEQ ID NO: 1, 3, 5, 7, 9, 11,13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 43; c) anucleic acid molecule which codes for a polypeptide whose sequence hasat least 50% identity to the sequences SEQ ID NO: 2, 4, 6, 8, 10, 12,14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42 or 44; d) anucleic acid molecule according to (a) to (c) which codes for a fragmentor an epitope of the sequences as shown in SEQ. ID NO: 2, 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 60,61 or 62; e) a nucleic acid molecule which codes for a polypeptide whichis recognized by a monoclonal antibody directed against a polypeptidewhich is encoded by the nucleic acid molecules as shown in (a) to (c);f) a nucleic acid molecule which hybridizes under stringent conditionswith a nucleic acid molecule as shown in (a) to (c); and g) a nucleicacid molecule which can be isolated from a DNA library using a nucleicacid molecule as shown in (a) to (c) or their part-fragments of at least15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt, asprobe under stringent hybridization conditions; or comprises acomplementary sequence thereof.
 4. The method according to claim 1,wherein the activity in lemma, palea, glume (anther primordium) isreduced.
 5. The method according to claim 1, wherein resistance isachieved by reducing expression of a polypeptide comprising two or moreArmadillo repeats.
 6. The method according to claim 1, wherein theendogenous sequence of a nucleic acid coding for an Armadillo repeatARM1 polypeptide is mutated.
 7. The method according to claim 1, whereinthe pathogens are selected from among the families Pucciniaceae,Mycosphaerellaceae and Hypocreaceae.
 8. The method according to claim 1,wherein a) the expression of the polypeptide is reduced; b) thestability of the polypeptide or of a mRNA molecule which corresponds tothis polypeptide is reduced; c) the activity of the polypeptide isreduced; d) the transcription of a gene coding for the polypeptide isreduced by the expression of an endogenous or artificial transcriptionfactor; or e) an exogenous factor which reduces the activity of thepolypeptide is added to food or to medium.
 9. The method claim 2,wherein the reduction in the activity of the polypeptide is achieved byapplying at least one method selected from the group consisting of: a)introducing a nucleic acid molecule coding for ribonucleic acidmolecules suitable for forming double-strand ribonucleic acid molecules(dsRNA), where the sense strand of the dsRNA molecule has at least 30%homology with the nucleic acid molecule characterized in claim 2, orcomprises a fragment of at least 17 base pairs, which has at least 50%homology with a nucleic acid molecule characterized in claim 2(a) or(b), b) introducing a nucleic acid molecule coding for an antisenseribonucleic acid molecule which has at least 30% homology with thenoncoding strand of a nucleic acid molecule characterized in claim 2 orcomprises a fragment of at least 15 base pairs with at least 50%homology with a noncoding strand of a nucleic acid moleculecharacterized in claim 2 (a) or (b), c) introducing a ribozyme whichspecifically cleaves the ribonucleic acid molecules encoded by one ofthe nucleic acid molecules mentioned in claim 2 or an expressioncassette which ensures the expression of such a ribozyme, d) introducingan antisense nucleic acid molecule as specified in b), in combinationwith a ribozyme or with an expression cassette which ensures theexpression of the ribozyme, e) introducing nucleic acid molecules codingfor sense ribonucleic acid molecules coding for a polypeptide which isencoded by a nucleic acid molecule characterized in claim 2, or forpolypeptides with at least 40% homology with the amino acid sequence ofa polypeptide encoded by the nucleic acid molecules mentioned in claim2, f) introducing a nucleic acid sequence coding for a dominant-negativepolypeptide suitable for suppressing the activity of the polypeptide, orintroducing an expression cassette which ensures the expression of thisnucleic acid sequence, g) introducing a factor which can specificallybind the polypeptide or the DNA or RNA molecules coding for thispolypeptide, or introducing an expression cassette which ensures theexpression of this factor, h) introducing a viral nucleic acid moleculewhich brings about a degradation of mRNA molecules which code for thepolypeptide, or introducing an expression cassette which ensures theexpression of this nucleic acid molecule. i) introducing a nucleic acidconstruct suitable for inducing a homologous recombination on genescoding for the polypeptide; and j) introducing one or more inactivatingmutations into one or more genes coding for the polypeptide.
 10. Themethod according to claim 9, comprising a) introducing, into a plantcell, a recombinant expression cassette comprising, in operable linkagewith a promoter which is active in plants, a nucleic acid sequence ascharacterized in claim 9 (a-i); b) regenerating the plant from the plantcell, and c) expressing the nucleic acid sequence in a sufficient amountand over a sufficient period of time to generate, or to increase, apathogen resistance in said plant.
 11. The method according to claim 10,wherein the promoter which is active in plants is a pathogen-induciblepromoter.
 12. The method according to claim 10, wherein the promoterwhich is active in plants is an epidermis- or mesophyll-specificpromoter.
 13. The method according to any of claims claim 1, wherein theactivity of a polypeptide coding for Bax inhibitor 1, ROR2, SnAP34and/or Lumenal Binding protein BiP is increased in a plant, plant organ,plant tissue or plant cell.
 14. The method according to claim 1, whereinthe activity of a polypeptide coding for RacB, CSL1, HvNaOX and/or MLOis decreased in a plant, plant organ, plant tissue or plant cell. 15.The method according to claim 13, wherein the Bax inhibitor 1 isexpressed under the control of a mesophyll- and/or root-specificpromoter.
 16. The method according to claim 1, wherein the pathogen isselected from among the species Puccinia triticina, Pucciniastriiformis, Mycosphaerella graminicola, Stagonospora nodorum, Fusariumgraminearum, Fusarium culmorum, Fusarium avenaceum, Fusarium poae orMicrodochium nivale.
 17. The method according to claim 1, wherein theplant is selected from the plant genera Hordeum, Avena, Secale,Triticum, Sorghum, Zea, Saccharum and Oryza.
 18. A nucleic acid moleculecoding for an Armadillo repeat ARM1 protein, comprising at least onenucleic acid molecule selected from the group consisting of: a) anucleic acid molecule which codes for at least one polypeptidecomprising the sequence shown in SEQ ID NO: 2; b) a nucleic acidmolecule which comprises at least one polynucleotide of the sequenceshown in SEQ ID NO: 1; c) a nucleic acid molecule which codes for apolypeptide whose sequences has at least 50% identity with the sequencesSEQ ID NO: 2; d) a nucleic acid molecule according to (a) to (c) whichcodes for a fragment or an epitope of the sequences as shown in SEQ. IDNO: 2; e) a nucleic acid molecule which codes for a polypeptide which isrecognized by a monoclonal antibody directed against a polypeptide whichis encoded by the nucleic acid molecules as shown in (a) to (c); f)nucleic acid molecule which hybridizes under stringent conditions with anucleic acid molecule as shown in (a) to (c); and g) a nucleic acidmolecule which can be isolated from a DNA library using a nucleic acidmolecule as shown in (a) to (c) or their part-fragments of at least 15nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt, as probeunder stringent hybridization conditions; or comprises a complementarysequence thereof; or a polypeptide comprising an amino acid sequenceencoded by the nucleic acid molecule; wherein the nucleic acid moleculedoes not comprise the sequence shown in SEQ ID NO.: 3, 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or
 43. 19. Anucleic acid construct comprising a nucleic acid molecule comprising atleast one fragment of a nucleic acid molecule which is antisense orsense relative to a nucleic acid molecule coding for Armadillo repeatARM1 polypeptide in operable linkage with a pathogen-inducible promoteror an epidermis- and/or mesophyll-specific promoter.
 20. The constructaccording to claim 19, wherein the Armadillo repeat ARM1 polypeptide isencoded by a nucleic acid molecule comprising a nucleic acid moleculeselected from the group consisting of a) a nucleic acid molecule whichcodes for a polypeptide comprising the sequence shown in SEQ ID NO: 2,4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,42, 44, 60, 61 or 62; b) a nucleic acid molecule which comprises atleast one polynucleotide of the sequence shown in SEQ ID NO: 1, 3, 5, 7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 43;c) a nucleic, acid molecule which codes for a polypeptide whose sequencehas at least 40% identity with the sequences SEQ ID NO: 2, 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42 or 44; d)a nucleic acid molecule according to (a) to (c) which codes for afragment or an epitope of the sequences as shown in SEQ. ID NO: 2, 4, 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42,44, 60, 61 or 62; e) a nucleic acid molecule which codes for apolypeptide which is recognized by a monoclonal antibody directedagainst a polypeptide which is encoded by the nucleic acid molecules asshown in (a) to (c); and f) a nucleic acid molecule which hybridizesunder stringent conditions with a nucleic acid molecule as shown in (a)to (c); and g) a nucleic acid molecule which can be isolated from a DNAlibrary using a nucleic acid molecule as shown in (a) to (c) or theirpart-fragments of at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100nt, 200 nt or 500 nt, as probe under stringent hybridization conditions;or which is complementary thereto.
 21. A double-stranded RNA nucleicacid molecule (dsRNA molecule), where the sense strand of said dsRNAmolecule has at least 30% homology with the nucleic acid molecule ascharacterized in claim 3, or comprises a fragment of at least 50 basepairs with at least 50% homology with the nucleic acid molecule ascharacterized in claim
 3. 22. The dsRNA molecule according to claim 21,wherein the two RNA strands are bonded covalently with one another. 23.A DNA expression cassette comprising a nucleic acid molecule which isessentially identical to a nucleic acid molecule as characterized inclaim 3, where said nucleic acid molecule is in sense orientationrelative to a promoter.
 24. A DNA expression cassette comprising anucleic acid molecule which is essentially identical to a nucleic acidmolecule as characterized in claim 3, where said nucleic acid moleculeis in antisense orientation relative to a promoter.
 25. A DNA expressioncassette comprising a nucleic acid sequence coding for a dsRNA moleculeaccording to claim 21, where said nucleic acid sequence is linked with apromoter.
 26. The DNA expression cassette according to claim 23, whereinthe nucleic acid sequence to be expressed is linked with a promoterwhich is functional in plants.
 27. The DNA expression cassette accordingto claim 26, wherein the promoter which is functional in plants is apathogen-inducible or an epidermis- and/or mesophyll-specific promoter.28. A vector comprising the expression cassette according to claim 23.29. A cell comprising a nucleic acid molecule as characterized in claim3, a dsRNA molecule where the sense strand of said dsRNA molecule has atleast 30% homology with the nucleic acid molecule as characterized inclaim 3 or comprises a fragment of at least 50 base pairs with at least50% homology with the nucleic acid molecule as characterized in claim 3,a DNA expression cassette comprising a nucleic acid molecule which isessentially identical to a nucleic acid molecule as characterized inclaim 3 where said nucleic acid molecule is in sense or antisenseorientation relative to a promoter, or a vector comprising theexpression cassette, or in which the endogenous activity of apolypeptide encoded by a nucleic acid molecule characterized as in claim3 is reduced or cancelled.
 30. A transgenic nonhuman organism comprisinga nucleic acid molecule as characterized in claim 3, a DNA expressioncassette comprising the nucleic acid molecule, a vector comprising theexpression cassette, or a cell comprising the nucleic acid molecule, theexpression cassette, or the vector.
 31. The transgenic nonhuman organismaccording to claim 30, which is a monocotyledonous organism.
 32. Thetransgenic nonhuman organism of claim 30, which has an increased flaxinhibitor 1 protein, an ROR2 and/or SnAP34 activity and/or a reducedRacB, CSL1 and/or HvRBOHF activity.
 33. The nonhuman organism of claim30, which has an increased Bax inhibitor 1, an ROBE and/or SnAP34activity and/or a reduced RacB, CSL1 and/or HvRBOHF activity inmesophyll cells and/or root cells.
 34. The organism according to claim30, selected among the species Hordeum vulgare (barley), Triticumaestivum (wheat), Triticum aestivum subsp. spelta (spelt), Triticale,Avena sativa (oats), Secale cereale (rye), Sorghum bicolor (millet), Zeamays (maize), Saccharum officinarum (sugar cane) or Oryza sativa (rice).35. (canceled)
 36. (canceled)
 37. A method for generating a plant whichis resistant to mesophyll-cell-penetrating pathogens comprisinggenerating a plant from the plant cell of claim 29, wherein the plant isresistant to mesophyll-cell-penetrating pathogens.
 38. Harvest,propagation material or composition comprising a nucleic acid moleculeas characterized in claim 3, a dsRNA molecule where the sense strand ofsaid dsRNA molecule has at least 30% homology with the nucleic acidmolecule as characterized in claim 3 or comprises a fragment of at least50 base pairs with at least 50% homology with the nucleic acid moleculeas characterized in claim 3, a DNA expression cassette comprising anucleic acid molecule which is essentially identical to a nucleic acidmolecule as characterized in claim 3 and where said nucleic acidmolecule is in sense or antisense orientation relative to a promoter, avector comprising the expression cassette, or a cell comprising thenucleic acid molecule, the expression cassette, or the vector.